Method for purifying and quantifying thrombin and its degradation polypeptides

ABSTRACT

Provided is a method for purifying α-thrombin and for quantifying α-thrombin and its degradation polypeptides in a liquid proteinatious solution. The method employs a one-step anion exchange chromatography method. The method allows purification and/or quantification of a homogenous post-translationally modified α-thrombin. The method can also be used for purification and/or quantification of β-thrombin.

FIELD OF THE INVENTION

Provided is a method that allows analyzing and quantifying α-thrombin,homogenously glycosylated α-thrombin and/or thrombin degradationpolypeptides in liquid proteinatious solutions. In particular, providedis an analytical and quantitative method that employs a singlechromatographic step. Also, provided is a method that allows efficientand robust purification of α-thrombin and/or homogenously glycosylatedα-thrombin without thrombin degradation polypeptides. The invention canalso be used for purification and/or quantification of β-thrombin.

BACKGROUND OF THE INVENTION

Thrombin is a serine protease which is widely used in clinicalapplications in several commercial products. It is a common component ofsurgical dressings, and has been used in combination with fibrinogen andother proteins in hemostatic systems such as fibrin glues, adhesives,and sealants. Fibrin sealants typically comprise a fibrinogen componentand a thrombin component. When both components are mixed (e.g. whenapplied to a bleeding wound or surgical incision) thrombin cleaves thefibrinogenpeptides off the fibrinogen thus allowing the latter togenerate insoluble fibrin polymers/sealant.

Concentrated (e.g. more than 500 IU/mL), purified thrombin in aqueousliquid form may display a reduction in activity during prolongedstorage, primarily as a result of autolysis. Assessment of thrombindegradation is thus an essential physico-chemical analytical tool fordetermining thrombin stability.

Mammalian α-thrombin is made up of two disulfide linked polypeptidechains A and B. The B chain is post-translationally modified (e.g. byglycosylation) and exhibits thrombin's proteolytic activity towardfibrinogen and other proteins. The α-thrombin can autolyze intoβ-thrombin, and γ-thrombin polypeptide derivatives, which can bepartially identified by Gel electrophoresis and Western Blot.

Thrombin autolysis is a major challenge in manufacturing and storing ofthrombin, especially at high concentrations. The methods known in theart for identifying thrombin degradation polypeptides (β-thrombin andγ-thrombin derivatives) are inadequate in that they either provideinsufficient separation between thrombin and its degradationpolypeptides, a denaturing separation and/or are labor intensive.Therefore, the quantitation is not accurate and/or possible.

Background art includes:

Boissel J P et al. “Covalent structures of beta and gamma autolyticderivatives of human alpha-thrombin”. J Biol Chem. 1984 May 10;259(9):5691-5697; Chang J Y. “The structures and proteolyticspecificities of autolysed human thrombin”. Biochem J. 1986 Dec. 15;240(3):797-802; Karlsson G. “Analysis of human alpha-thrombin byhydrophobic interaction high-performance liquid chromatography”. ProteinExpr Purif 2003 January; 27(1):171-174; European Patent No. EP 0443724;and WO 2004/103519.

Boissel et. al. describes the use of CEX-HPLC followed by RP-HPLCanalysis to separate the different thrombin degradation polypeptides.Chang describes HPLC analysis of pure thrombin fractions separated bySEC chromatography and further analyzed using RP-HPLC. The above methodshave the shortcoming of requiring at least two separation steps forquantification and separation of thrombin from other proteins.

Karlsson describes hydrophobic interaction chromatography (HIC) toseparate thrombin degradation products.

European Patent No. EP 0443724 discloses a method for preparing a viralsafe thrombin, however, the method is denaturing and shows no separationbetween the different thrombin degradation products or between thedifferent α-thrombin post-translational variants.

WO 2004/103519 discloses methods for the separation of charged moleculessuch a proteins according to their isoelectric points (pI's) andincludes the systems and buffering compositions employed for isolatingcharged molecules.

There remains an unmet need for analytical methods for quantifyingα-thrombin or β-thrombin; and for the purification of active, intactα-thrombin or of β-thrombin from proteinatious solutions which overcomethe above defects of the art.

SUMMARY OF THE INVENTION

Provided is a one-step chromatographic method for quantifying α-thrombinand/or homogenous post-translationally modified α-thrombin in asolution, the solution comprising the α-thrombin and at least one of anα-thrombin degradation polypeptide (β-thrombin and/or γ-thrombinpolypeptide), post-translationally modified α-thrombin species oranother protein.

Also, provided are methods for purifying α-thrombin from proteinatioussolutions by providing good separation of intact e.g. non-degraded,functional, active α-thrombin and/or homogenous post-translationallymodified α-thrombin, the solutions comprising the α-thrombin and atleast one of an α-thrombin degradation polypeptide (β-thrombin and/orγ-thrombin polypeptide), post-translationally modified α-thrombinspecies or another protein.

Also, provided are methods for purifying a homogenous α-thrombinglycoform from a solution comprising heterogeneous glycosylatedα-thrombin species.

Also, provided are methods for purifying and/or quantifying β-thrombinin a solution comprising the β-thrombin and at least one of α-thrombine.g. post-translationally modified α-thrombin species, γ-thrombin oranother protein.

As used herein, the term “at least one of” is both conjunctive anddisjunctive in operation. For example, the expressions “at least one ofA, B or C” means: A alone, B alone, C alone, A and B together, A and Ctogether, B and C together, or A, B and C together.

Homogenous post-translationally modified α-thrombin can be homogenouslyglycosylated α-thrombin or homogenously glycosylated and homogenouslysialylated α-thrombin.

A homogenous α-thrombin glycoform according to the instant applicationcan be a “homogenously glycosylated α-thrombin” or “a homogenouslyglycosylated and homogenously sialylated α-thrombin species”.

Typically, a glycoform is an isoform of a protein that differs only withrespect to the number and/or type of attached glycans orpolysaccharides. Glycoproteins often consist of a number of differentglycans, with alterations in the attached saccharides.

Often, the terms “glycan” and “polysaccharide” refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein. Glycans can behomo- or hetero-polymers of monosaccharide residues, and can be linearor branched. The glycans can carry saccharides with or without negativecharges.

The methods comprise: contacting the solution with an Anion Exchanger.The methods allow getting a robust and reproducible performance,providing highly purified and active α-thrombin and/or homogenouspost-translationally modified α-thrombin; and accurate quantification ofα-thrombin, homogenous post-translationally modified α-thrombin and/orits degradation polypeptides. The method also enables the quantificationand/or purification of β-thrombin stand-alone.

In one aspect, provided is a method for purifying α-thrombin from asolution comprising the α-thrombin and at least one of an α-thrombindegradation polypeptide or another protein, the method comprising thesteps of: 1-contacting the solution with an anion exchanger;2-separating the α-thrombin from the at least one of the α-thrombindegradation polypeptide (e.g. from β-thrombin and/or γ-thrombinpolypeptide) and/or the another protein by an anion exchangechromatography (AEX) using differential elution conditions; and3-collecting an α-thrombin fraction, thereby obtaining purifiedα-thrombin.

In some embodiments, the method comprises separating the α-thrombin fromthe at least one of the α-thrombin degradation polypeptide (e.g.β-thrombin and/or γ-thrombin polypeptide) and the another protein by ananion exchange chromatography (AEX) using differential elutionconditions.

In one embodiment, the α-thrombin is from a human blood or plasmasource. In another embodiment, the α-thrombin is from a recombinantsource.

The term “separating” used herein typically refers to isolating aspecific compound from a solution comprising the specific compound andother compounds.

In one aspect, provided is a method for purifying homogeneouslyglycosylated α-thrombin from a solution comprising heterogeneouslyglycosylated α-thrombin, the method comprising the steps of:1-contacting the solution with an anion exchanger; 2-separating thehomogeneously glycosylated α-thrombin from the heterogeneouslyglycosylated α-thrombin by an anion exchange chromatography (AEX) usingdifferential elution conditions; and 3-collecting a homogeneouslyglycosylated α-thrombin fraction, thereby obtaining purifiedhomogeneously glycosylated α-thrombin.

In one aspect, provided is a method for purifying homogeneouslyglycosylated α-thrombin from a solution comprising heterogeneouslyglycosylated α-thrombin and at least one of an α-thrombin degradationpolypeptide or another protein. In another aspect, provided is a methodfor purifying homogeneously glycosylated α-thrombin from a solutioncomprising at least one of heterogeneously glycosylated α-thrombin, anα-thrombin degradation polypeptide or another protein. The methodcomprising the steps of: 1-contacting the solution with an anionexchanger; 2-separating the homogeneously glycosylated α-thrombin by ananion exchange chromatography (AEX) using differential elutionconditions; and 3-collecting a homogeneously glycosylated α-thrombinfraction, thereby obtaining purified homogeneously glycosylatedα-thrombin.

In some embodiments after step 1—the contacting step, a washing step iscarried out using an isocratic buffer/solution.

In one embodiment of the invention, the method comprises the steps of:loading the thrombin containing solution to an anion exchanger; washingwith an isocratic solution; discarding the washed fraction; and elutinga desired α-thrombin fraction using a non-isocratic solution such as apH gradient. Use of an isocratic solution typically relates to the useof a constant-composition mobile phase in liquid chromatography.

“A desired α-thrombin fraction” typically refers to any α-thrombinpresent in a solution for which purification and/or quantification isintended for, including, for example, homogeneous post-translationallymodified α-thrombin e.g. homogeneously glycosylated α-thrombin orhomogeneously glycosylated and homogeneously sialylated α-thrombin.

In one aspect, provided is a method for purifying a homogenousα-thrombin glycoform from a solution comprising heterogeneousglycosylated α-thrombin species, the method comprising the steps of:

contacting the solution with an anion exchanger;

separating the homogenous α-thrombin glycoform from the heterogeneousspecies by anion exchange chromatography using differential elutionconditions, and

collecting a homogenous α-thrombin glycoform fraction,

thereby obtaining purified homogenous α-thrombin glycoform.

In one embodiment, the method also comprises the step of quantifying thepurified homogenous α-thrombin glycoform.

In another aspect, provided is a one-step or single step chromatographicmethod for quantifying α-thrombin in a solution comprising theα-thrombin and at least one of an α-thrombin degradation polypeptide oranother protein, the method comprising the steps of: separating theα-thrombin from the at least one of the α-thrombin degradationpolypeptide or the another protein on anion exchange chromatography bydifferential elution conditions; collecting an α-thrombin fraction; andquantifying the α-thrombin. In another aspect, provided is a one-step orsingle step chromatographic method for quantifying α-thrombin in asolution comprising the α-thrombin and at least one of an α-thrombindegradation polypeptide or another protein, the method comprising thesteps of: contacting the solution with an anion exchanger; separatingthe α-thrombin from the at least one of the α-thrombin degradationpolypeptide or the another protein on anion exchange chromatography bydifferential elution conditions such as a pH gradient; collecting anα-thrombin fraction; and quantifying the α-thrombin.

In some embodiments, the α-thrombin is from a mammalian e.g. human orpig plasma source or a recombinant protein.

The chromatographic methods disclosed herein can be carried out usingall techniques known to the person skilled in the art. For example, aHigh-Performance Liquid Chromatography device; a Fast Protein LiquidChromatography (FPLC) and/or a stand-alone column with or without aconnected detector can be employed.

In one embodiment, an Anion Exchange High-Performance LiquidChromatography method is used. High-performance liquid chromatography(HPLC; also referred to as high-pressure liquid chromatography), istypically a technique that relies on pumps to pass a pressurized liquidsolvent containing the sample mixture through a column filled with asolid adsorbent material. Each component in the sample interactsslightly differently with the adsorbent material, leading to theseparation of the components. HPLC is distinguished from traditional(“low pressure”) liquid chromatography because operational pressures aresignificantly higher (50-350 bar). Some models of mechanical pumps in aHPLC instrument can mix multiple solvents together in ratios changing intime, generating a composition gradient in the mobile phase. Variousdetectors are in common use, such as Ultra Violet (UV), photodiode array(PDA) or mass spectrometry. The detection can be carried out using UVabsorbance detector at 190-400 nm (A_(190nm)-A_(400 nm)). In oneembodiment, when amines are included in the elution buffer, absorbanceis measured at about A_(280nm).

Typically, a chromatographic separation e.g. an HPLC run consists atleast of the following steps: an equilibrated column is contacted e.g.loaded with a sample/mixture (“Loading”). After loading a washing stepcan be carried out. Following this step, the separated components areeluted from the column. This can be carried out isocratically (withoutchanging the buffer composition as compared to the loading and/orequilibration steps) or through a gradient (changing at least one of thebuffer characteristic, e.g. salt concentration, polarity, pH). In oneembodiment, elution is carried out using a linear gradient. In the nextstep, the column can be regenerated (“Column regeneration”), meaningthat the remaining components are given additional time at the highestconcentration of the changed characteristic (salt concentration,polarity, pH) in order to elute from the column any remaining material.Regeneration can alternatively be carried out by changing other buffercharacteristics (not changed during the elution step). The last step(“Column equilibration”) can be an equilibration step, to allow thecolumn to return to the original state in which the column is suitablefor an additional use. The described steps can alternatively be carriedout using an FPLC device and/or a stand-alone column. Chromatographicseparation is well known in the art as described in Hidayat Ullah Khan(2012). The Role of Ion Exchange Chromatography in Purification andCharacterization of Molecules, Ion Exchange Technologies, Chapter 14,331-334.

Advantageously, the methods according to the invention provide good peakseparation of intact α-thrombin from its degradation polypeptides and/orfrom other proteins in the thrombin solution. Typically, inchromatographic methods “good separation”/“good peak separation” isconsidered an efficient separation of the components, in which the peaksdetected, as representative of elution of the components, do notoverlap; that is, the detector response returns to the base line levelbetween the peaks. The term “good peak separation” is also meant toinclude “sufficient separation” in which a clear distinction between theeluting peaks appears, however, the detector response does not fullyreturn to the base line level between the peaks.

Separation/resolution efficacy can be visually evaluated. Alternativelyor in addition, the resolution (Rs), the extent to which achromatographic column separates components from each other, can bemathematically defined: resolution is the difference between the peakretention times of a selected peak and the peak preceding it multipliedby a constant of 1.18, then divided by the sum of the peak widths at 50%of peak height. The term “retention time” refers to the interval betweenthe instant of injection and detection of the peak apex (the most upperpoint of the peak) as representative of elution.

Generally, a resolution level of equal to or above 2 is considered asgood separation of the component and allows good quantitation of thepeak. A resolution of equal to or above 1.5 (and lower than 2) isconsidered as “sufficient separation” which enables separation and/orquantitation.

In one embodiment of the methods, the resolution between α-thrombinpeaks and its degradation polypeptides is in the range of about 1.5 toabout 8.

In one embodiment of the methods, the resolution between α-thrombinpeaks and other proteins in the thrombin solution is higher than 8.

In one embodiment of the methods, the resolution between the differentα-thrombin species peaks is in the range of about 1.5 to about 8.

In one embodiment, the resolution between the different α-thrombindegradation polypeptides (β-thrombin and γ-thrombin) is lower than 1.5such as equal to 0. In one embodiment, β-thrombin and γ-thrombin elutein the same peak. In another embodiment, a certain β-thrombin formelutes in a separate peak e.g. the resolution between β-thrombin andother components in the solution is about 1.5 to about 8. Accordingly,in one aspect, the invention also provides a method for purifyingβ-thrombin from a solution comprising the β-thrombin and at least one ofα-thrombin, β-thrombin or another protein, the method comprising thesteps of:

contacting the solution with an anion exchanger; separating theβ-thrombin from the at least one of the α-thrombin, γ-thrombin and/oranother protein by anion exchange chromatography using differentialelution conditions; and collecting a β-thrombin fraction, therebyobtaining purified β-thrombin.

The term “β-thrombin fraction” typically refers to the fractioncollected following elution of the loaded anion exchanger (e.g. loadedcolumn) with a buffer under differential elution conditions.

In another aspect, the invention provides a one-step chromatographicmethod for quantifying β-thrombin in a solution comprising theβ-thrombin and at least one of α-thrombin, γ-thrombin or anotherprotein, the method comprising the steps of: contacting the solutionwith an anion exchanger; separating the β-thrombin from the at least oneof the α-thrombin, γ-thrombin and/or the another protein on anionexchange chromatography by differential elution conditions; andquantifying the β-thrombin.

In some embodiments, the method further includes identifying theseparated β-thrombin, α-thrombin and/or γ-thrombin containing fractions.In some embodiments, the method further includes quantifying α-thrombinand/or γ-thrombin.

In some embodiments, the method comprises separating the β-thrombin fromthe at least one of the α-thrombin, γ-thrombin and another protein byanion exchange chromatography using differential elution conditions.

In some embodiments, the chromatographic method is an anion exchangeHigh-Performance Liquid Chromatography method. In some embodiments, thedifferential elution conditions comprise a pH gradient e.g. generated byusing an eluent comprising of an amine or a mixture of amines. In someembodiments, the anion exchanger is made of non-porous particles.

In another aspect, the invention provides a purified β-thrombinobtainable by the methods of the invention; an isolated β-thrombin; anda formulation/kit comprising the purified/isolated β-thrombin asdescribed herein.

In some embodiments, the method allows separating and collectinghomogenous post-translationally modified α-thrombin fractions. In someembodiments, the homogenous post-translationally modification ishomogenous glycosylation. In some embodiments, the homogenouspost-translationally modification is homogenous glycosylation andsialylation. In some embodiments, the separated/collected α-thrombinfraction is a homogenous glycosylated α-thrombin. In some embodiments,the homogenous post-translationally modified α-thrombin is representedby a single glycoform. In some embodiments, the separated/collectedα-thrombin glycoform is homogeneously glycosylated and/or homogeneouslysialylated. In some embodiments, the homogeneity of the isolated,separated and/or collected post-translationally modified α-thrombin e.g.the homogenous α-thrombin glycoform is a level of at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or at least100% identity. E.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or lessthan 100%, including any range between the disclosed percentages such as50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%,50-99%, 50-100%, 55-60%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%,55-95%, 55-99%, 55-100%, 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%,60-95%, 60-99%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%,65-99%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%,70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-100%, 80-85%,80-90%, 80-95%, 80-99%, 80-100%, 85-90%, 85-95%, 85-99%, 85-100%,90-95%, 90-99%, 90-100%, 95-99%, 95-100% identity. In yet anotherembodiment, the α-thrombin is un-modified, e.g. non-glycosylated.

In some embodiments, the proteinatious solution includes at least one ofanother protein, an α-thrombin degradation polypeptide (for example,β-thrombin polypeptide and/or γ-thrombin polypeptide), an α-thrombinthat is not post-translationally modified (an unmodified α-thrombin), orpost-translationally modified α-thrombin.

In some embodiments, the solution includes a mixture of unmodified andpost-translationally modified α-thrombin.

In some embodiments, the solution includes heterogeneouspost-translationally modified α-thrombin including different glycoformspecies of α-thrombin.

In some embodiments, the solution includes another protein or a proteinfragment, which may be, for example, a protein that was added to thesolution. In some embodiments, the protein is, for example, human serumalbumin (HSA). In some embodiments of the method, the solution includesat least one of α-thrombin degradation polypeptide or another protein.

In some embodiments of the method, the solution includes at least one ofα-thrombin degradation polypeptide or HSA.

In some embodiments of the method, the solution includes at least one ofα-thrombin degradation polypeptide and HSA.

In some embodiments, the method comprises the step of: loading thesolution onto an anion exchange column. In some embodiments, the methodcomprises contacting the solution with an anion exchanger in batch-wiseform.

As used herein, “batch method”, “batch-wise”, and “batch form” generallyrefer to a technique in which a solution is contacted with a resin,typically in a single stage adsorption procedure. “A single stageadsorption procedure” refers to a procedure wherein all the componentsof the purification process (e.g. the resin and the solution) areincubated together e.g. in a stirred tank, batch reactor or a vessel,and the adsorption is carried out in a continuous manner. Theresin-bound fraction can then be collected by an additional step ofcentrifugation and/or filtration.

In some embodiments, prior to contacting e.g. loading, theexchanger/column is equilibrated to a pH of 10.5 to about pH 7.0 (e.g. apH of 9.1). The equilibration can be carried out using buffer or bufferssuitable for equilibrating the exchanger at a pH of 10.5 to about pH 7.0(e.g. a pH of 9.1).

In one embodiment, the buffer comprises a mixture of amines. In someembodiments, the amines mixture used for equilibration includespiperazine, triethanolamine, bis-tris propane, 1-methylpiperazine,bicine, bis-tris, diethanolamine, diethylamine, 1-histidine, imidazole,pyridine, tricine, triethanolamine, and/or tris.

In some embodiments, the amines mixture used for equilibration consistsof piperazine, triethanolamine, bis-tris propane and 1-methylpiperazine.In some embodiments, the concentration of the amines in theequilibration buffer is in the range of about 1 to about 100 mM e.g. inthe range of about 10 to about 20 mM or about 20 mM.

The flow rate during contacting e.g. loading can be in the range ofabout 0.1 to about 1.4 mL/minute. In some embodiments, the conditionsfor allowing separation between degradation polypeptides of α-thrombin,α-thrombin, homogenous α-thrombin glycoform and/or another proteinincludes applying differential elution conditions such as subjecting theanion exchanger/column to pH gradient conditions for elution.

In some embodiments, the differential elution conditions compriseapplying a pH gradient e.g. stepwise or continuous (e.g. linear).Typically, a “continuous gradient” is defined as a gradient in which theeluent composition is changed gradually, continuously and constantlywhile the “stepwise gradient” includes instant changes in the eluentcomposition.

In some embodiments, the gradient length is in the range of 5 minutes to100 minutes or 5 minutes to 60 minutes. In another embodiment, thegradient length is higher than 25 minutes e.g. higher than 30 or higherthan 35 minutes. In some embodiments, the gradient length is in therange of higher than 25 minutes to 35 minutes or in the range of higherthan 25 minutes to 30 minutes.

In one embodiment, elution is carried out with the same buffer used forequilibration of the anion exchanger.

In some embodiments, the linear pH gradient is from about pH 10.5 toabout pH 2.0 such as in the range of about 9.1 to about pH 3.4.

In some embodiments, the pH gradient is generated using an eluentcomprising of an amine or a mixture of amines. In some embodiments, thelinear pH gradient is generated using an eluent buffer comprising amixture of amines. In some embodiments, the amines mixture used duringthe differential elution conditions includes piperazine,triethanolamine, bis-tris propane, 1-methylpiperazine, bicine, bis-tris,diethanolamine, diethylamine, 1-histidine, imidazole, pyridine, tricine,triethanolamine, and/or tris. In some embodiments, the amines mixtureused during the differential elution conditions consists of piperazine,triethanolamine, bis-tris propane and 1-methylpiperazine. In someembodiments, the concentration of each amine in the buffer is in therange of about 1 to about 100 mM. In some embodiments, the concentrationof each amine in the buffer is about 20 mM.

In some embodiments, the linear pH gradient is generated using twoeluent buffers comprising a mixture of amines. In some embodiments, thelinear pH gradient is generated using two eluent buffers comprising thesame mixture of amines. Typically, the pH of the eluting buffer isdependent on the ratio between Buffer A and B during the elution. Insome embodiments, Buffer A has a pH of about 9.1 and Buffer B has a pHof about 3.4, and the concentration of Buffer A decreases from about 40%to about 60% and Buffer B increases from about 60% to about 40%. In someembodiments, Buffer A has a pH of about 9.1 and Buffer B has a pH ofabout 3.4, and the concentration of Buffer A decreases from about 100%to about 0% and Buffer B increases from about 0% to about 100%. In someembodiments, Buffer A has a pH of about 9.1 and Buffer B has a pH ofabout 3.4, and the concentration of Buffer A decreases from about 90% toabout 0% and Buffer B increases from about 10% to about 100%. In someembodiments, the increment of % Buffer B per minute is in the range ofabout 0.1% to about 10% or in the range of about 3.5% to about 4.5%. Insome embodiments, the increment of % Buffer B per minute is selectedfrom the group consisting of about 3.5%, 3.75%, 4%, 4.25%, or 4.5%. Insome embodiments, the increment of % Buffer B per minute is about 3.5%.

In some embodiments, the elution conditions comprise a flow rate ofabout 0.1 to about 1.4 mL/minute, or about 0.25 to 1.0 mL/minute, or 0.5mL/minute to 0.8 mL/minute, or about 0.8 to about 1.0 mL/minute. In someembodiments, the elution conditions comprise a flow rate of about 1mL/minute.

In some embodiments, the elution conditions comprise the followingsteps: from 90% to 100% Buffer B at a linear increase/slope of about0.1% to about 10%, about 0.5% to about 10%, or about 3.5% to about 4.5%Buffer B per minute.

In some embodiments, the elution conditions comprise the followingsteps: from 0% to 100% Buffer B at a linear increase/slope of about 0.1%to about 10%, about 0.5% to about 10%, or about 3.5% to about 4.5%Buffer B per minute.

In some embodiments, the elution conditions comprise the followingsteps: from 90% to 100% Buffer B at a linear increase/slope of about3.5% Buffer B per minute.

In some embodiments, the elution conditions comprise the followingsteps: from 0% to 100% Buffer B at a linear increase/slope of about 3.5%Buffer B per minute.

In some embodiments of the methods, the anion exchanger is a weak or astrong anion exchanger. In some embodiments of the method, the anionexchanger consists of quaternary ammonium positively charged groups. Insome embodiments of the method, the anion exchanger is based on about 1to about 1000 μm e.g. 5 μm polymer beads. In some embodiments of themethod, the polymer beads consist of poly(styrene/divinyl/benzene). Insome embodiments of the method, the anion exchanger consists ofnon-porous or porous particles e.g. the pores of the particles are inthe range of about 120 to 1000 Ångstrom (Å). In some embodiments of themethod, the anion exchanger consists of non-porous particles. In someembodiments of the method, the anion exchanger consists of monodisperseparticles.

In some embodiments of the method, an anion exchange column having atleast one of the following characteristics is used: a width in the rangeof 1.7 to 10 mm (e.g. 4.6 mm), and a length in the range of 10 to 250 mm(e.g. 250 mm).

In some embodiments of the method, an anion exchange column having awidth in the range of 1.7 to 10 mm (e.g. 4.6 mm) and a length in therange of 10 to 250 mm (e.g. 250 mm) is used.

In some embodiments of the methods, the method consists of one stepchromatographic method e.g. one type of chromatographic method withoutadditional chromatographic and/or separation step(s).

In some embodiments, the purification method is carried out by an anionexchange High-Performance Liquid Chromatography, a Fast Protein LiquidChromatography (FPLC) and/or by a stand-alone column with or without aconnected detector.

In some embodiments, the method is for analytical purposes.

In certain embodiments, provided herein is a purified α-thrombinobtainable by the methods provided herein.

In another aspect, provided herein is an isolated homogenouspost-translationally modified α-thrombin. In some embodiments, theα-thrombin is from a mammalian plasma source e.g. from a human or pigplasma source. In another aspect, provided herein is an isolatedhomogenous post-translationally modified α-thrombin from mammalian bloodor plasma source.

In some embodiments, the post-translationally modification isglycosylation. In some embodiments, the post-translationallymodification is glycosylation and sialylation. In some embodiments, thehomogenous post-translationally modified α-thrombin is represented by asingle/particular glycoform. In some embodiments, the α-thrombinglycoform is further sialylated. In some embodiments, the α-thrombinglycoform is homogenously sialylated. In some embodiments, the isolatedhomogenous post-translationally modified α-thrombin is homogeneouslyglycosylated α-thrombin. In some embodiments, the isolated homogenouspost-translationally modified α-thrombin is represented by oneparticular glycoform. In some embodiments, the isolated homogenouspost-translationally modified α-thrombin is homogeneously sialylatedα-thrombin.

In yet another aspect, provided herein is a formulation comprising apurified α-thrombin or an isolated homogeneous post-translationallymodified α-thrombin as disclosed herein. In some embodiments, thepurified α-thrombin or an isolated homogeneous post-translationallymodified α-thrombin is obtained by the methods disclosed herein. In someembodiments of the formulation, the α-thrombin is from mammalian plasmasource. In some embodiments, the α-thrombin is from blood or plasmasource. In some embodiments, the formulation comprises apharmaceutically acceptable carrier or diluent. The formulationdisclosed herein can be frozen or lyophilized.

In another aspect, provided herein is a method of providing a hemostatictreatment, sealing, graft fixation, wound healing and/or anastomosis, toa surface in a subject, comprising applying to the surface a formulationcomprising the purified α-thrombin, the homogeneous post-translationallymodified α-thrombin or the β-thrombin. The formulation can be appliedwith a solution comprising fibrinogen. The surface can be a bleeding ora non-bleeding site. The subject may be a human subject.

In another aspect, the invention relates to the use of a formulationcomprising an isolated homogenous post-translationally modifiedα-thrombin, a purified α-thrombin or β-thrombin as disclosed hereinabovefor hemostatic treatment, sealing, graft fixation, wound healing,anti-adhesion and/or anastomosis.

In another aspect, provided is a kit comprising a container such as anampoule, a vial and/or a syringe which includes the purified α-thrombin,the homogeneous post-translationally modified α-thrombin or theβ-thrombin as disclosed hereinabove; and optionally an applicationdevice and/or instructions for use.

In another aspect, provided is a kit comprising a container comprisingan isolated homogenous post-translationally modified α-thrombin or apurified homogenous α-thrombin glycoform as disclosed hereinabove as afirst component.

In some embodiments, the kit comprises a container comprising gelatine.g. as a second component. The kit may further include fibrinogen. Insome embodiments, the kit comprises a container comprising fibrinogene.g. as a second component. The kit may include at least one containerand at least one label. Suitable containers include, for example,ampoules, vials, syringes and tubes. The containers can be made of, forexample, glass, metal or plastic.

These and other aspects and embodiments of the invention will becomeevident upon reference to the following detailed description of theinvention and the figures.

All embodiments disclosed herein relating to purification and/orquantification of α-thrombin also relate to purification and/orquantification of β-thrombin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a zoom-in view of a representative chromatogram in theregion of the eluting peaks obtained using a Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) of several samples:HSA, thrombin solution, formulated thrombin and water. The samplesinjected were: a) 30 μL thrombin solution; b) 100 μL formulatedthrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL, HPLC grade water as ablank sample.

In all Figs. the sample depictions is shown from top to bottom based onthe beginning of the chromatogram, the sample injected (from top tobottom) is listed on the chromatogram. The runs of the different samplesare shown in one figure as stacked overlays. In all graphs the γ-axis islabeled as Absorbance Unit (AU) and the x-axis is labeled as minutes.

FIG. 2 shows a zoom-in view of a representative chromatogram in theregion of the eluting peaks obtained using Anion ExchangeHigh-Performance Liquid Chromatography (AEX-HPLC) and elution with alinear NaCl salt gradient at pH 8.0. The samples injected were: a) 30 μLthrombin solution; b) 100 μL formulated thrombin; c) 100 μL 5 mg/ml HSA;and d) 100 μL Buffer A as a blank sample.

FIG. 3 shows a representative chromatogram obtained for differentsamples injected into the AEX-HPLC and eluted using a linear NaCl saltgradient at pH 6.0. The samples injected were: a) 30 μL thrombinsolution; b) 100 μL formulated thrombin; c) 100 μL 5 mg/ml HSA; and d)100 μL Buffer A as a blank sample.

FIG. 4 shows a zoom-in view of a representative chromatogram in theregion of the eluting peaks obtained using AEX-HPLC with a linear NaClsalt gradient at pH 7.5. The samples injected were: a) 30 μL thrombinsolution; b) 100 μL formulated thrombin; c) 100 μL 5 mg/ml HSA; and d)100 μL Buffer A as a blank sample.

FIG. 5 shows a representative chromatogram in the region of the elutingpeaks obtained using AEX-HPLC with a linear NaNO₃ salt gradient at pH8.0. The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; and c) 100 μL 5 mg/ml HSA.

FIG. 6 shows a representative chromatogram of the samples injectedobtained using AEX-HPLC with a linear pH gradient between pH 9.1 to pH3.4. The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL Buffer A as ablank sample. The elution was carried out with amine based buffers.

FIG. 7 is a zoom-in view of the thrombin eluting region in thechromatogram shown in FIG. 6.

FIG. 8 shows a zoom-in view of the chromatograms obtained using AEX-HPLCand elution at a linear pH gradient at different flow rates: 0.25, 0.5,0.75, and 1 mL/min. The injected sample was 30 μL thrombin solution foreach tested flow rate.

FIG. 9 shows the effect of the linear gradient slope on theseparation/resolution (evaluated by visual inspection). Differentincrements of % Buffer B per minute were evaluated: 4.5%, 4.25%, 4%,3.75%, and 3.5%. The injected sample was 30 μL thrombin solution foreach tested increment.

FIG. 10 shows overlaid chromatograms of α, β and γ thrombin standards,thrombin solution and Buffer A as a blank sample obtained using AEX-HPLCat a flow rate of 1.0 mL/min. Different thrombin peaks were identifiedfor the thrombin solution by comparison to the thrombin commercialstandards.

FIG. 11 shows different thrombin peaks separated from a thrombinsolution using an AEX-HPLC linear pH gradient between 100% Buffer A to100% Buffer B in a slope of 3.5% B per minute. The eluted peaks werecollected and further identified using Western Blot as a qualitativetool.

FIG. 12 shows a chromatogram with different thrombin species resolved byHPLC-AEX. A thrombin solution subjected to sialic acid removal and athrombin solution without treatment were injected. The results show thatsialic acid removal affects the charge of the present thrombin specieswhich in turn affects the elution profile resulting in an overall shiftof the peaks to the left side of the chromatogram (as compared to anun-treated thrombin solution).

FIG. 13 shows a full length chromatogram of an injected thrombin sampleobtained using an AEX-HPLC linear pH gradient, with an increment of 3.5%Buffer B per minute, and flow conditions of 1.0 mL/min. The results showcomplete separation between human serum albumin, several α-thrombinpeaks corresponding to different homogenously post-translationallymodified α-thrombin species and acetyltryptophan.

FIG. 14 shows a zoom-in view of the α-thrombin species and degradationpolypeptides eluting region.

DETAILED DESCRIPTION OF THE INVENTION

The method provided herein is based, in part, on the discovery that ahomogeneous e.g. with respect to the post-translational modification(e.g. glycosylation and/or sialylation level), intact α-thrombin may beisolated/purified from a heterogeneous protein solution by utilizingAnion Exchange Chromatography (AEX). Also, the method according to theinvention is based on the discovery that α-thrombin or β-thrombin can beisolated/purified from a solution comprising other proteins e.g. astabilizer such as human serum albumin and/or bovine serum albumin.

It was surprisingly found that the method according to the inventionenables a high resolution purification and/or quantification ofα-thrombin or β-thrombin in the presence of high amounts of otherproteins relative to thrombin concentration in the solution.

More particularly, the method according to the invention enables topurify and/or quantify different homogenous post-translationallymodified α-thrombin species (e.g. homogenous α-thrombin glycoforms) inhigh resolution from a heterogeneous solution comprising high amounts ofother proteins e.g. stabilizers such as human serum albumin, bovineserum albumin and the like.

In some embodiments, the other proteins, e.g. serum albumin, are presentin the solution at a concentration of about 0.4 to about 50 mg/ml e.g.about 5 to about 6.5 mg/ml. In some embodiments, the thrombinconcentration in the solution is in the range of about 100 to about10000 IU/ml e.g. about 800 to about 1200 IU/ml or about 0.3 mg/ml. Insome embodiments, the ratio of thrombin (IU) to other proteins (mg) isin the range of about 1:10 to about 1:40 or about 1:14 to about 1:27.

In particular, provided herein is a one-step chromatographic method forpurification and/or quantification of α-thrombin from a thrombincomprising solution by providing good peak separation of intact,post-translationally modified α-thrombin from its degradationpolypeptides, and other proteins in a thrombin formulation. Also,provided herein are tools for separating between α-thrombin, itsdegradation polypeptides and other proteins (e.g. HSA) in a thrombinsolution/formulation.

“Intact α-thrombin” refers, for example, to an undamaged, non-degradedand/or functional form of α-thrombin.

Hitherto, thrombin was purified and analyzed using reverse phasechromatography, hydrophobic interaction chromatography, cation exchangechromatography and/or SDS-PAGE and Western Blot.

Provided herein is a method for purifying α-thrombin from a solutioncomprising the α-thrombin and at least one of an α-thrombin degradationpolypeptide (e.g. β-thrombin and/or γ-thrombin polypeptides) or anotherprotein, the method comprising the steps of: contacting the solutionwith an anion exchanger; separating the α-thrombin from at least one ofthe α-thrombin degradation polypeptide or the another protein by ananion exchange chromatography using differential elution conditions e.g.pH gradient; and collecting an α-thrombin fraction, thereby obtainingpurified α-thrombin.

Also, provided herein is a method for purifying a homogenouspost-translationally modified α-thrombin species (e.g. homogenousα-thrombin glycoform) from a solution comprising heterogeneouspost-translationally modified α-thrombin species (e.g. heterogeneousglycosylated α-thrombin species), and optionally at least one of anα-thrombin degradation polypeptide or another protein; the methodcomprising the steps of: contacting the solution with an anionexchanger; separating the homogenous post-translationally modifiedα-thrombin species from the other α-thrombin post-translationallymodified species; and optionally from the α-thrombin degradationpolypeptide and/or the another protein; by differential elutionconditions e.g. pH gradient, and collecting a homogenouspost-translationally modified α-thrombin fraction, thereby obtainingpurified homogenous post-translationally modified α-thrombin species.

The terms “purifying”, “to purify” and the like refer to removing,isolating, or separating α-thrombin (e.g. a homogeneous,post-translationally modified α-thrombin) or β-thrombin from a solutioncomprising it. The α-thrombin containing solution may also compriseα-thrombin degradation polypeptide (β-thrombin and/or γ-thrombin),another protein and/or other α-thrombin post-translationally modifiedspecies. The β-thrombin containing solution may also compriseα-thrombin, γ-thrombin, another protein and/or α-thrombinpost-translationally modified species.

The term “contacting” refers to any type of a combining action whichbrings the solution into sufficiently close contact with the anionexchanger comprising the positively charged groups, in a manner that abinding interaction will occur between the positively charged groups andany binding partner, e.g. α-thrombin or β-thrombin, present in thesolution. The solution can be incubated with the anion exchanger for asufficient period of time, e.g. 1 min or more, which allows contactingand/or binding between the positively charged groups and the α-thrombinor β-thrombin.

The term “α-thrombin fraction” typically refers to the fractioncollected following elution of the loaded anion exchanger (e.g. loadedcolumn) with a buffer under differential elution conditions. In oneembodiment, the collected α-thrombin fraction consists of onlyα-thrombin. In another embodiment, the collected α-thrombin fractionconsists of one homogenous α-thrombin species. In another embodiment,the collected α-thrombin fraction consists of homogenous α-thrombinglycoform fraction.

The term “purified α-thrombin”, typically, refers to an α-thrombinpreparation obtained following isolation of the α-thrombin fromα-thrombin degradation polypeptides and/or another protein present inthe starting thrombin comprising solution using an anion exchangechromatography method. The term “purified α-thrombin”, as used herein,also refers to a homogeneous post-translationally modified α-thrombine.g. homogeneously glycosylated and/or sialylated α-thrombin preparationobtained following isolation of the homogeneously post-translationallymodified α-thrombin from heterogeneously post-translationally modifiedα-thrombin solution using an anion exchange chromatography method. Theterm “purified homogenous α-thrombin glycofrom”, typically, refer to ahomogenous α-thrombin glycoform preparation obtained following isolationof the α-thrombin glycoform from α-thrombin degradation polypeptides,heterogeneous glycosylated α-thrombin species and/or another proteinpresent in the starting thrombin comprising solution using an anionexchange chromatography method.

In one embodiment, the purified α-thrombin is an intact protein withoutdegradation polypeptides.

In one embodiment, the purified α-thrombin is a homogenouspost-translationally modified α-thrombin species. In another embodiment,the purified α-thrombin is an unmodified α-thrombin species. In anotherembodiment, the purified α-thrombin is a homogenous α-thrombinglycoform.

A purified α-thrombin preparation may consist of a homogenouspost-translationally modified species isolated from a solutioncomprising various post-translationally modified α-thrombin. Thestarting thrombin solution may also comprise unmodified α-thrombinspecies.

The isolated post-translationally modified α-thrombin may beglycosylated or glycosylated and sialylated. The glycosylation and/orsialylation degree may vary between the different species. The antennacan be branched at varying degrees from di-antennary to penta-antennary.The sialic acid can be any of the derivatives of the neuraminic acid(5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), likeN-acetylneuraminic acid or N-glycolylneuraminic acid.

“α-thrombin” may include unmodified α-thrombin, homogeneous orheterogeneous α-thrombin, homogenous post-translationally modifiedα-thrombin, for example, homogenously glycosylated α-thrombin orhomogenously glycosylated and homogenously sialylated α-thrombin orhomogeneously sialylated α-thrombin; and heterogeneouslypost-translationally modified α-thrombin e.g. heterogeneouslyglycosylated or glycosylated and sialylated α-thrombin. The α-thrombinmay be from a mammalian blood and/or plasma source e.g. human, bovineplasma or pig plasma source or from a recombinant source.

In some embodiments, the “another protein”/“other proteins” is humanserum albumin (HSA) or any other protein included in a thrombinformulation e.g. for stabilization of the formulation. The “anotherprotein” is different from α, β and γ-thrombin. The “another protein”may be numerous proteins which can be found in the blood or plasma suchas prothrombin, immunoglobulins, HSA and others. The another protein maybe a protein fragment. In some embodiments, the another protein is astabilizer such as human serum albumin and/or bovine serum albumin.

The term “anion exchange chromatography” refers to a separationtechnique wherein molecules are separated based on their net charge.Anion exchangers are named for their ability to attract or bind anionsor negatively charged particles. Anion exchangers are well known in theart (Practical Protein Chromatography edited by Kenney and Fowell Volume11; Chapter 16, 249-258; Humana Press, 1992). In anion exchangers, theresin is positively charged and a molecule will bind if the buffer pH ishigher than the protein's isoelectric point. The term “isoelectricpoint” refers to the pH wherein a molecule carries no net charge. In amedium with a pH below the isoelectric point, the molecule carries a netpositive charge, above it the molecule carries a net negative charge.The terms “anion exchanger” and “anion exchange matrix” are used hereininterchangeably.

The terms “support” and “resin” as used herein include a carrier, or anymatrix used to attach, immobilize, carry, or stabilize the positivelycharged groups. Supports are well known in the art as described inHermanson G T, Mallia A K and Smith P K 1992 “Immobilization AffinityLigand Techniques” pp. 1-45 Academic Press, Inc. San Diego, USA.

The support for carrying out the method of the invention can be made ofany material which is capable of binding a molecule comprisingpositively charged groups i.e. a molecule comprising chemical groupswhich carry a positive charge. Solid supports include, but are notlimited to, matrices, columns, coverslips, chromatographic materials,filters, microscope slides, test tubes, vials, bottles, ELISA supports,glass or plastic surfaces, chromatographic membranes, sheets, particles,beads, including magnetic beads, gels, powders, fibers, and the like.

In one embodiment of the invention, the support is in the form of achromatographically utilizable material. In another embodiment of theinvention, the support is in the form of a chromatographic membrane. Thesupport can be composed of a hydrophilic material such as agarose,sepharose, acrylic beads, cellulose, controlled pore glass, silica gels,dextranes; hydrophobic material; or an organic artificial/syntheticpolymer such as materials based on polyacrylamides or polystyrens.Typical materials/polymers are commercially available under the tradenames Sephacryl® (Pharmacia, Sweden), Ultragel® (Biosepara, France)TSK-Gel Toyopearl® (Toso Corp., Japan), HEMA (Alltech Ass. (Deer-field,Ill., USA), Eupergit® (Rohm Pharma, Darmstadt, Germany). Also materialsbased on azlactones (3M, St. Paul, Minn., USA). In one embodiment, thesupport is composed of Agarose® or Sepharose®. These materials arecommercially available. Typically, anion-exchange resin oranion-exchange polymer is an insoluble matrix (or support structure)normally in the form of small beads, fabricated from an organic polymersubstrate.

In some embodiments of the method, the beads are at a size of about 1 toabout 1000 μm or e.g. 5 μm. The beads can be non-porous or porousparticles. The beads can be monodisperse (i.e. substantially homogenousin size) particles. In one embodiment, the beads used are in the rangeof 1.7 μm to 10 μm.

The anion exchanger can be a weak or a strong anion exchanger. A weakanion exchanger generally refers to an exchanger which is comprised of aweak base, while a strong anion exchanger generally refers to anexchanger which is comprised of a strong base that is able to sustainits charge over a wider pH range.

In some embodiments of the method, the positively charged groups areselected from the group consisting of ammonium, alkyl ammonium,dialkylammonium, trialkyl ammonium, quaternary ammonium, alkyl groups,H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, amino functional group, and a combinationthereof.

Resin beads can be suspended in an appropriate medium and the resultingslurry can be used e.g. in a chromatographic column referred to hereinas “column purification”. Alternatively, the column can be purchased ina pre-packed form.

“Column purification” and “column chromatography” generally refer to atechnique in which a solution (the mobile phase) is allowed to travelthrough a column comprising a packed resin at a certain flow rate, andan individual component or a number of components are adsorbed by theresin (the stationary phase) i.e. by the chromatographic material. Theun-bound material can be collected from the other side of the columnafter the mixture has passed through it. By using certain elutionconditions it is possible to alter the bond between the differentcompounds and the stationary phase, thereby leading to the elution of aspecific, purified compound from the column, one at a time. Columnpurification is well known in the art as described in Practical ProteinChromatography edited by Kenney and Fowell Volume 11; Chapter 16,249-258; Humana Press, 1992.

Typically, a slurry of resin, is poured into the column. After itsettles, the column is pre-equilibrated in buffer before the proteinmixture/solution is applied. Alternatively, a pre-packed column can bepurchased. Unbound proteins appear in the flow-through and/or insubsequent buffer washes. Proteins that bind to the resin are retainedand can be eluted by: salt or pH/polarity adjustment. The term “unboundmaterial”/“un-bound fraction” typically refers to the fraction discardedfollowing washing of the loaded column e.g. with the same buffer usedfor equilibration and/or the buffer used for loading the thrombincontaining solution onto the column (“binding buffer”). “A non-isocraticsolution” is used as elution conditions. A “non-isocratic solution”typically refers to, e.g. a solution and/or a condition that isdifferent from the solution and/or condition used to load, wash and/orequilibrate the column; and/or to a solution that is different from asolution used in a previous step. Elution conditions employ a shift inthe composition of the mobile phase so the factors binding environmentcreated by the binding buffer is changed.

Generally, equilibration is carried out until pH and/or conductivityand/or UV readings are stabilized. In one embodiment, equilibration iscarried out with >5 column volumes of buffer. In another embodiment,equilibration is carried out with 1 to 5 column volumes of buffer.

The term “elution conditions” refers to the use of a non-isocraticcondition e.g. a solution and/or condition different from the solutionand/or condition used to load and/or equilibrate the column; and/ordifferent from the solution used in a previous step. The solution and/orconditions used at the starting point and/or at the end point of theelution step (e.g. the gradient elution) may be identical to thesolution and/or conditions used during the washing, loading, and/orregeneration steps. The term “elution conditions” may also refer to agradient elution during which salt concentration and/or changes inpH/polarity occurs. The elution conditions are such that the proteinsand degradation polypeptides are separated and eluted differentially.The method according to the invention comprises at least one elutionstep with a non-isocratic solution. Elution conditions, typicallyinvolve an increase in salt concentration and/or changes in pH/polarity.It was found herein that using a pH gradient for elution is efficient.

In some embodiments of the methods, the method consists of onechromatography step i.e. a single chromatography step.

Typically, the term “one-step chromatographic method” or “onechromatography step” or “one-step anion exchange chromatography” refersto a method enabling the purification and/or quantitation of theα-thrombin; homogeneous or heterogeneous α-thrombin; homogenouslypost-translationally modified α-thrombin, for example, homogenouslyglycosylated α-thrombin or homogenously glycosylated and homogenouslysialylated α-thrombin; unmodified α-thrombin; heterogeneouslypost-translationally modified α-thrombin e.g. heterogeneouslyglycosylated or glycosylated and sialylated α-thrombin and/or thrombindegradation polypeptides; or β-thrombin that is carried out by the anionexchanger directly on a sample material without additionalchromatographic and/or separation step(s).

In one embodiment of the invention, column purification is utilized. Inanother embodiment, an Anion Exchange High-Performance LiquidChromatography method is used. The column may be regenerated afterelution of the solution ingredients for repetitive use. The total runtime from loading to regeneration can be in the range of 30 to 120minutes e.g. about 46, 55 minutes. In one embodiment, the gradientseparation takes 28.6 minutes.

Separation according to the invention is carried out by employingdifferential elution conditions. The term “differential elutionconditions” refers to conditions that allow separation of α-thrombinfrom its degradation polypeptides and/or from another protein;separation of a homogenous post-translationally modified α-thrombinspecies from thrombin degradation polypeptides, another protein and/orheterogeneous post-translationally modified α-thrombin species;separation of a homogenous α-thrombin glycoform from heterogeneouspost-translationally modified α-thrombin species e.g. glycosylatedα-thrombin species; separation of a homogenous α-thrombin glycoform fromat least one of α-thrombin degradation polypeptides, another protein orheterogeneous post-translationally modified α-thrombin species; and/orseparation of β-thrombin from α-thrombin, γ-thrombin and/or anotherprotein.

The elution conditions may involve alterations in the salt concentrationand/or in the pH of the elution buffer. In one embodiment, thedifferential elution conditions comprise alterations in the pH e.g. a pHgradient. In one embodiment, the resins used according to the inventionare adequate to work at a pH range according to the invention. In oneembodiment, the resins are suitable to be subjected to organic materials(such as methanol and/or acetonitrile).

The column volume can be in the range of about 0.03 to about 53 mL. Inone embodiment of the invention, the column volume is about 4.1 mL e.g.4.15 mL. In another embodiment, most of the peaks are collected withinone column volume.

In an additional embodiment, the method is an analytical method, e.g.physico-chemical analytical method, and can be carried out as a one-stepchromatographic method.

In some embodiments, the method further includes identifying theseparated α-thrombin, β-thrombin and/or γ-thrombin containing fractions.

In some embodiments, the method further includes identifying thedifferent post-translationally modified α-thrombin. The differentglycosylations can be analyzed using Mass Spectroscopy, capillaryelectrophoresis, by using different HPLC methods or by any other methodsknown in the art.

In one embodiment, the different fractions/peaks are visually identifiedafter injecting the sample set. Typically, the peaks profile is robustand therefore each peak can be easily identified. In another embodiment,the different thrombin peaks are identified by injecting into the HPLCα, β and γ thrombin standards, and identifying the correlating peaks ofthe thrombin solution. In another embodiment, the different thrombinpeaks are identified by Western Blot analysis by running the elutedpeaks against α, β and γ thrombin standards and/or based on the knownmolecular size of α-, β- and γ-thrombin. Provided herein is a one-stepchromatographic method for quantifying α-thrombin in a solutioncomprising the α-thrombin and at least one of an α-thrombin degradationpolypeptide or another protein, the method comprising the steps of:separating the α-thrombin from the at least one of the α-thrombindegradation polypeptide or the another protein on anion exchangechromatography by differential elution conditions; collecting anα-thrombin fraction; and quantifying the α-thrombin.

Provided herein is a one-step chromatographic method for quantifying ahomogenous post-translationally modified α-thrombin (e.g. homogenousglycoform) in a solution comprising heterogeneous post-translationallymodified α-thrombin and optionally at least one of an α-thrombindegradation polypeptide or another protein, the method comprising thesteps of: separating the homogenous post-translationally modifiedα-thrombin from the solution on anion exchange chromatography bydifferential elution conditions; collecting the homogenouspost-translationally modified α-thrombin fraction; and quantifying thehomogenous post-translationally modified α-thrombin. Provided herein isa one-step chromatographic method for quantifying α-thrombin in asolution comprising the α-thrombin and at least one of an α-thrombindegradation polypeptide or another protein, the method comprising thesteps of: contacting the solution with an anion exchanger; separatingthe α-thrombin from the at least one of the α-thrombin degradationpolypeptide and/or the another protein on anion exchange chromatographyby differential elution conditions; and quantifying the α-thrombin.

In some embodiment, the method comprises separating the α-thrombin fromthe at least one of the α-thrombin degradation polypeptide and theanother protein on anion exchange chromatography by differential elutionconditions.

In some embodiments, the method further includes quantifying one or moredegradation polypeptides e.g. β-thrombin and/or γ-thrombin polypeptides.

Also, provided herein is a one-step chromatographic method forquantifying homogenous post-translationally modified α-thrombin in asolution comprising heterogeneous post-translationally modifiedα-thrombin; and optionally at least one of an α-thrombin degradationpolypeptide or another protein, the method comprising the steps of:contacting the solution with an anion exchanger; separating thehomogenous post-translationally modified α-thrombin from theheterogeneous post-translationally modified α-thrombin; and optionallyfrom the at least one of the α-thrombin degradation polypeptide and/orthe another protein; on anion exchange chromatography by differentialelution conditions; and quantifying the homogenous post-translationallymodified α-thrombin. In one embodiment, the solution comprises at leastone of α-thrombin degradation polypeptide β-thrombin and/or γ-thrombin);and/or another protein.

In some embodiments, the solution further comprises at least one of anα-thrombin degradation polypeptide or another protein, and the methodincludes separating the homogenous post-translationally modifiedα-thrombin also from the at least one of the α-thrombin degradationpolypeptide and/or the another protein. In some embodiments, the methodincludes separating the homogenous post-translationally modifiedα-thrombin from the at least one of the α-thrombin degradationpolypeptide and the another protein.

Quantification can be carried out, for example, by calculating theintegration e.g. by measuring the area under the peak of a chromatogram.The peaks can be quantitated either by integration of the peak andcomparing the peak area to the total area eluted or by evaluating thepeak height. The area or height can be translated into absolute numbersif a standard is used or the relative peak area can be evaluated.

In some embodiments of the methods, the separating step includesapplying differential elution conditions. In some embodiments theelution conditions include applying a pH gradient. In some embodimentsthe elution conditions include applying a linear pH gradient. Typically,a linear pH gradient is defined as a gradient which gradually andequally changes the pH over time. In some embodiments, the pH gradientis from about pH 9.1 to about pH 3.4. In some embodiments the linear pHgradient is generated using an eluent comprising an amine or a mixtureof amines. In some embodiments, the eluent comprises a mixture ofamines. In some embodiments, the amine based buffer comprisespiperazine, triethanolamine, bis-tris propane, 1-methylpiperazine and amixture thereof. In some embodiments, the concentration of each amine inthe buffer is in the range of about 1 to about 100 mM e.g. in the rangeof about 10 to about 20 mM or about 20 mM. Buffers with similarcharacteristics, suitable for the creation of a pH gradient, can be usede.g. phosphate buffers at different pH values. Alternative compounds,not listed herein can be used to build a buffer system suitable for theelution of thrombin from an anion exchanger.

The results show that AEX-HPLC and elution using a linear gradientbetween pH 9.1 to pH 3.4 lead to good resolution between HSA,acetyltryptophan, α-thrombin degradation polypeptides and α-thrombin.Accordingly, in one embodiment, a linear pH gradient elution stepbetween pH 9.1 to pH 3.4 is used as differential elution conditions. Inone embodiment, the HPLC comprises a loading step of 5 minutes, at aflow rate of 0.80 mL/min; a linear pH gradient elution step of 20minutes, at a flow rate of 0.80 mL/min, the linear pH gradient isgenerated by using two eluent buffers comprising the same mixture ofamines, the concentration of Buffer A decreases from 90% to 0% andBuffer B increases from 10% to 100%, the increment of Buffer B is 4.5%per minute. In another embodiment, the HPLC comprises a columnequilibration step of 15 minutes, at a flow rate of 0.80 mL/min. Inanother embodiment, the HPLC comprises a column regeneration step of 5minutes, at a flow rate of 0.80 mL/min.

In some embodiments, the temperature during the elution step is in therange of about 10° C. to about 50° C. e.g. about 25° C.

In some embodiments, the flow rate during the linear pH gradient elutionstep is 0.25, 0.5, 0.75, and 1 mL/min. The results show that theresolution between α-thrombin and its degradation polypeptides increaseswith increasing flow rates and that the best resolution was achieved ata flow rate of 1.0 mL/min. Accordingly, in one embodiment, the flow rateduring the linear pH gradient elution step is higher than 0.75 mL/mine.g. about 1.0 mL/min.

The results show that eluting the proteins from the column with a widerpH range leads to a better separation between the peaks. Accordingly, inone embodiment, a linear pH gradient elution step is generated by usingtwo eluent buffers comprising the same mixture and concentrations ofamines, the gradient concentration of Buffer A decreases from 100% to 0%and Buffer B increases from 0% to 100%, the increment of Buffer B isabout 4.5% per minute. In such an embodiment, the linear pH gradientelution step can be about 22 minutes.

The total run time from loading the thrombin solution onto the columnand up to column regeneration step (e.g. including a loading steps, alinear gradient elution step, a column regeneration step and a columnequilibration step) can be in the range of 30 to 120 minutes e.g. in therange of 46 to 61 minutes such as about 46, 51, 56, and 61 minutes totalrun time, and the elution step can be in the range of 20 to 35 minutese.g. about 20, 25, 30 and 35 minutes. The results show that at 56 and 61minutes total run times (a gradient elution step length of 30 and 35minutes), an additional peak eluting in a region distinct to thethrombin peaks was separated as compared to the shorter run times.Accordingly, in one embodiment, a linear gradient elution step of higherthan 25 minutes is carried out.

The results show that elution with a linear pH slope gradient of 4.5%,4.25%, 4%, 3.75%, and 3.5% per minute was efficacious in separation ofthe different thrombin peaks with an increment of 3.5% having the bestseparation profile. The slope gradient can be impacted by the incrementof the percentage of Buffer B per minute when more than one buffer isused as an eluent buffer. Typically, a lower increase of the percentageof Buffer B per minute results in a shallower slope as compared to ahigher increase of the percentage of Buffer B per minute, therebyaffecting the elution profile of the proteins. Accordingly, in someembodiments, the elution is carried out at a linear pH gradient between100% Buffer A to 100% Buffer B with a slope of 3.5-4.5% Buffer B perminute e.g. at a slope of 3.5%.

In some embodiments, the starting solution (to be purified and/orquantified) comprising the α-thrombin further comprises another protein,and substantially lacks degradation polypeptides (e.g. the solutioncontains less than 10% w/w β-thrombin and/or γ-thrombin relative to thetotal thrombin amount).

In some embodiments, the starting solution comprising the α-thrombinfurther comprises degradation polypeptides e.g. β-thrombin and/orγ-thrombin. In some embodiments, the starting solution comprising theα-thrombin further comprises degradation polypeptides without theanother protein.

In some embodiments, the starting solution comprising the α-thrombinfurther comprises degradation polypeptides (e.g. β-thrombin and/orγ-thrombin), and another protein.

In some embodiments, the starting solution comprising the β-thrombinfurther comprises α-thrombin and lacks γ-thrombin and/or anotherprotein. In some embodiments, the starting solution comprising theβ-thrombin further comprises γ-thrombin and lacks α-thrombin and/oranother protein. In some embodiments, the starting solution comprisingthe β-thrombin further comprises another protein and lacks α-thrombinand/or γ-thrombin. In some embodiments, the starting solution comprisingthe β-thrombin further comprises α-thrombin, and γ-thrombin and lacksanother protein. In some embodiments, the starting solution comprisingthe β-thrombin further comprises α-thrombin, and another protein andlacks γ-thrombin. In some embodiments, the starting solution comprisingthe β-thrombin further comprises γ-thrombin and another protein andlacks α-thrombin. In some embodiments, the starting solution comprisingthe β-thrombin further comprises α-thrombin, γ-thrombin and anotherprotein.

The starting solution may comprise heterogeneous post-translationallymodified α-thrombin and/or un-modified α-thrombin. In some embodiments,the starting solution comprises another protein. In some embodiments,the starting solution lacks another protein.

The thrombin concentration in the solution may be in the range of fromabout 2 to about 10000 IU/mL, from about 100 to about 10000 IU/mL, fromabout 2 to about 4000 IU/mL, about 800 to about 3000 IU/mL or about 800to about 1200 IU/mL and the total protein concentration may be in therange of about 0.3 to about 55 mg/ml, about 0.3 to about 10 mg/ml orabout 1 to about 7 mg/ml. In some embodiments, 1000 IU/ml equals 0.3mg/ml. The solution may be at a pH in the range of about 6.9 to about7.1, and may comprise between 5.0 to 6.5 mg/mL human serum albumin (HSA)and/or other stabilizers such as acetyltryptophan.

A solution comprising α-thrombin may be a solution comprising a thrombinformulation or formulated thrombin (e.g. a thrombin solution comprisingexcipients and/or stabilizers) e.g. a drug product, e.g. with a thrombinactivity in the range of 800-1200 IU/ml, a total protein concentrationof about 5.7-6.5 mg/ml, and 5.0 to 6.5 mg/ml human serum albumin (HSA)at pH 6.9-7.1. The thrombin formulation may include other stabilizerse.g. acetyltryptophan. In one embodiment, the HSA used for theformulation of thrombin includes a stabilizer, acetyltryptophan.

As used herein the terms “excipient” refers to an inert substance whichis added to the pharmaceutical composition. Examples of excipientsinclude, but are not limited to, human albumin, mannitol, sodium acetateand water for injection. The human albumin in the solution can be in therange of from about 2 to about 8 mg/ml. Mannitol can be in theconcentration range of from about 15 to about 25 mg/ml. Sodium acetatecan be also added in the solution in the range of from about 2 to about3 mg/ml.

In one embodiment, the thrombin solution comprises about 3000 IU/mlthrombin, a total protein concentration of about 1 mg/ml, 20 mM sodiumacetate at pH 6.9-7.1. In another embodiment, the thrombin formulationcomprises thrombin in the range of 800-1200 IU/ml, a total proteinconcentration of about 5.7-6.5 mg/ml, and 5.0 to 6.5 mg/ml human serumalbumin (HSA) at pH 6.9-7.1.

The solution may comprise calcium chloride. Calcium chlorideconcentration in the solution can be in the range of from about 2 toabout 6.2 mg/ml, or in the range of from about 5.6 to about 6.2 mg/ml,such as in the concentration of 5.88 mg/ml.

Thrombin clotting activity can be measured directly, for example, byEuropean Pharmacopeia Assay (0903/1997) procedure, by measuringmigration length on a slanted surface (or drop test model), and/or byany other method known in the art.

Thrombin activity may be determined using a coagulation analyzer with amechanical endpoint detection system to detect clot formation, such asthe Diagnostica Stago ST4 Coagulation Analyzer, or a device thatmeasures changes in turbidity due to fibrin clot formation.

Another method by which thrombin activity can be measured is using achromogenic or fluorogenic peptide substrate for thrombin. Oftentimes,in this method, solubilized thrombin is combined with an excess ofchromogenic or fluorogenic substrate. Thrombin will cleave the substratereleasing a chromophore or fluorophore which can be monitored in aspectrophotometer or fluorimeter. Examples of chromogenic or fluorogenicsubstrates include, β-Ala-Gly-Arg-p-nitroanilide diacetate andZ-Gly-Pro-Arg-AMC [Z=Benzyloxycarbonyl; AMC=7-amino-4-methylcoumarin],respectively. The rate of released chromophore or fluorophore can becorrelated to the activity of thrombin.

Thrombin can be prepared from a blood composition. The blood compositioncan be whole blood or blood fractions, i.e. a fraction of whole bloodsuch as plasma. The origin of the thrombin can be autologous whereby itwould be manufactured from the patient's own blood, from pooled blood orfractions. The thrombin solution can be prepared from plasma of humanbeings or mammals. In one embodiment, the thrombin is prepared byrecombinant methods in prokaryotic cells.

In one embodiment, the thrombin solution can be formulated as a sterilesolution, pH 6.8-7.2, which contains highly purified human thrombin. Thethrombin formulation can contain: human thrombin (800-1200 IU/mL),calcium chloride, human albumin, mannitol, sodium acetate and water forinjection. In one embodiment, thrombin is manufactured bychromatographic purification of prothrombin from cryo-poor plasmafollowed by activation with calcium chloride e.g. as described in U.S.Pat. No. 5,143,838, which is incorporated herein by reference.

In another aspect, provided herein is a one-step analytical method forquantifying α-thrombin in formulated thrombin (e.g. a drug product)including the α-thrombin and another protein (e.g. human serum albumin),the method comprising the steps of: contacting the formulated thrombinwith an anion exchanger; separating the α-thrombin from the anotherprotein on anion exchange chromatography by differential elutionconditions; and quantifying the α-thrombin. In some embodiments, thedifferential elution conditions comprise a pH gradient e.g. generated byusing an eluent comprising of an amine or a mixture of amines. In someembodiments, an anion exchange High-Performance Liquid Chromatographymethod is used. In some embodiments, the anion exchanger is made ofnon-porous particles. In some embodiments, the formulated thrombinfurther comprises undesired α-thrombin degradation polypeptides(β-thrombin and/or γ-thrombin polypeptides), and the method includesseparating the α-thrombin from the degradation polypeptides. In someembodiments, the formulated thrombin does not contain degradationpolypeptides.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, steps or components but do not preclude the addition of one ormore additional features, steps, components or groups thereof.

When a numerical value is preceded by the term “about”, the term “about”is intended to indicate ±10%.

“Thrombin” or “thrombin polypeptide” is a mammalian serine proteasewhich is part of the blood coagulation cascade and converts fibrinogeninto insoluble strands of fibrin, as well as catalyzing othercoagulation-related reactions. In humans, prothrombin is encoded by theF2 gene, and the resulting polypeptide is proteolytically cleaved in thecoagulation cascade by Factor Xa with a co-factor (FVa) or other serineproteases to generate thrombin. Thrombin serves, inter alia, as anactive component in several hemostasis products. For example, fibrinsealants typically comprise a fibrinogen component and a thrombincomponent. When both components are mixed (e.g. when applied to ableeding wound) thrombin cleaves fibrinogen and a fibrin polymer isformed which has hemostatic characteristics. Fibrin sealant is typicallya blood product obtained from either commercial sources or some regionalblood transfusion centers. Components that are commonly used in thepreparation of fibrin glues are fibrinogen, thrombin, Factor VIII,Factor XIII, fibronectin, vitronectin and von Willebrand factor (vWF).Fibrin sealant is typically formed by an enzymatic reaction involvinginter alia, fibrinogen, thrombin and Factor XIII. The terms “fibrinsealant” and “fibrin glue” are interchangeable.

Human thrombin is a 295 amino acid protein composed of two polypeptidechains, A and B, joined by a disulfide bond. The B chain of α-thrombinis responsible for thrombin's proteolytic activity on fibrinogen andother proteins and for its autolytic activity leading to the β-thrombinand γ-thrombin degradation polypeptides. Cleavage of the B-chain at theArg106-Tyr107 bond yields a 70 amino acid B1 fragment and the 188 aminoacid β-thrombin (B2) form. The γ-thrombin is generated by furthercleavage of the β-thrombin B2-chain at the Lys190-Gly191 bond.Typically, these proteolyzed forms of thrombin have reduced ability tocovert fibrinogen into insoluble strands of fibrin than intactα-thrombin.

The human α-thrombin B chain is further post-translationally modifiede.g. by glycosylation, possibly resulting in a more potent and/or stableform of thrombin as compared to an unmodified form and/or as compared toother form of post-translational modification (as indicated by RicardoJ. Sola and Kai Griebenow. “Glycosylation of Therapeutic Proteins: AnEffective Strategy to Optimize Efficacy”. BioDrugs. 2010; 24(1): 9-21for other glycosylated proteins). Mature α-thrombin has a singleN-linked glycosylation site on its “heavy chain”. Sialic acid, alsoreferred to as neuraminic acid, is critical to glycoproteinbioavailability, function, stability, and metabolism. The glycosylatedform of α-thrombin (in mature, natural human α-thrombin, amino acidresidue N416) may be further sialylated with from 1 to 5 sialic acidresidues. Accordingly, α-thrombin may contain different sialylationdegrees/levels e.g. α-thrombin may vary in the amount ofN-acetylneuraminic acid (NANA) residues (sialic acid) in theglycosylation site. The degree of sialylation may influence proteinpotency and stability. Typically, the higher the sialylation level, thehigher the potency and the higher the stability.

The results show that separating α-thrombin by anion exchangechromatography enables the separation of numerous α-thrombin peakscontaining different amounts of NANA. The results show that treatment ofthrombin with N-acetylneuraminidase, an enzyme capable of removing theNANA residues from the terminal end of glycans, affects the elutionprofile of thrombin resulting in an overall shift of the peaks to theleft side of the chromatogram (as compared to an un-treated thrombin).Accordingly, the method of the invention can be used to purify and/orquantify different α-thrombin glycoforms e.g. having differences in NANAcontent. In one embodiment, the method according to the invention can beused to purify/isolate different homogenous α-thrombin speciescontaining a substantially identical profile of NANA using AEX-HPLC. Inanother embodiment, the method according to the invention can be used topurify/isolate homogenously post-translational modified α-thrombin froma proteinatious solution and/or from a solution comprisingheterogeneously post-translational modified α-thrombin.

“Post-translational modification” is a step in protein biosynthesis.Proteins are created by ribosomes translating mRNA into polypeptidechains. The polypeptide chains undergo post-translational modifications,e.g. cutting, folding, and other processes, before they mature into thefinal protein product.

After translation, the post-translational modification of amino acidsextends the range of functions of the protein by attaching it to otherbiochemical functional groups, changing the chemical nature of an aminoacid, or making structural changes (e.g. formation of disulfidebridges). Modifications can be glycosylation, phosphorylation,ubiquitination, methylation, nitrosylation, acetylation, lipidation.Typically, modifications control the behavior of a protein e.g.activating or inactivating an enzyme.

Typically, glycosylation has a significant effect on protein folding,conformation, distribution, stability and activity. Glycosylationincludes addition of a sugar-moiety to proteins that ranges from simplemonosaccharide modifications of nuclear transcription factors to highlycomplex branched polysaccharide changes. Phosphorylation plays acritical role in the regulation of many cellular processes includingcell cycle, growth, apoptosis and signal transduction pathways.Methylation, the transfer of one-carbon methyl groups to nitrogen oroxygen (N- and O-methylation, respectively) to amino acid side chainsincreases the hydrophobicity of a protein and can neutralize a negativeamino acid charge when bound to carboxylic acids. Ubiquitination,ubiquitin is an 8-kDa polypeptide consisting of 76 amino acids that isappended to the Îμ-NH2 of lysine in a target protein via the C-terminalglycine of ubiquitin. Polyubiquitinated proteins are recognized by the26S proteasome that catalyzes the degradation of the ubiquitinatedprotein and the recycling of ubiquitin. Methylation is a well-knownmechanism of epigenetic regulation, as histone methylation anddemethylation influences the availability of DNA for transcription.Amino acid residues can be conjugated to a single methyl group ormultiple methyl groups to increase the effects of modification.

An “unmodified α-thrombin” refers to α-thrombin that did not undergopost-translational modifications e.g. non-glycosylated and/or thereforenon-sialylated α-thrombin.

A “homogeneous, post-translationally modified α-thrombin” refers to asubstantially identical form of α-thrombin e.g. with regards of theglycosylation and/or the sialylation level. The homogeneity between thedifferent α-thrombin molecules is expressed by having the samepost-translationally modification, e.g. same glycosylation, however eachthrombin molecule can possess different levels and/or forms of othermodifications. The α-thrombin can be a glycosylated and/or sialylatedform of α-thrombin. In one embodiment, the homogeneous α-thrombin ishomogeneously glycosylated. In another embodiment, the homogeneousα-thrombin is homogeneously sialylated. The glycosylated α-thrombin canhave from 0 to 5 sialic acid residues. In some embodiments, thehomogeneous post-translationally modified α-thrombin is a sialylatedα-thrombin having 1, 2, 3, 4 or 5 sialic acid residues.

As used herein, the different/heterogeneous post-translationallymodified α-thrombin populations of α-thrombin and unmodified α-thrombinis also known as “different α-thrombin species”. The heterogeneouspost-translationally modified α-thrombin may possess differentglycosylation and/or sialylation forms.

As used herein, an “α-thrombin glycoform” refers to a homogenouslyglycosylated and/or sialylated α-thrombin species.

In some embodiments, the α-thrombin that is prepared using the methoddescribed herein is homogeneous to a level of at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or at least 100%identity. E.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or less than100%, including any range between the disclosed percentages such as50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%,50-99%, 50-100%, 55-60%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%,55-95%, 55-99%, 55-100%, 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%,60-95%, 60-99%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%,65-99%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%,70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-100%, 80-85%,80-90%, 80-95%, 80-99%, 80-100%, 85-90%, 85-95%, 85-99%, 85-100%,90-95%, 90-99%, 90-100%, 95-99%, 95-100% identity.

The present disclosure provides a method of isolating a homogeneouspopulation/species of α-thrombin e.g. a homogenouslypost-translationally modified α-thrombin. Furthermore, provided is apurified homogeneous population of α-thrombin and a formulationcomprising the isolated homogenous post-translationally modifiedα-thrombin; and a pharmaceutically acceptable carrier or diluent.

In yet another aspect, provided herein is a formulation comprisingpurified α-thrombin or an isolated homogeneous post-translationallymodified α-thrombin as disclosed herein. In some embodiments, thepurified α-thrombin or the isolated homogeneous post-translationallymodified α-thrombin is obtained by the methods disclosed herein. In someembodiments, the purified α-thrombin or the isolated homogeneouspost-translationally modified α-thrombin is obtainable by the methodsdisclosed herein. In some embodiments of the formulation, the α-thrombinis from mammalian plasma source. In some embodiments, the formulationcomprises a pharmaceutically acceptable carrier or diluent. Theformulation disclosed herein can be frozen or lyophilized.

The formulation comprising the purified α-thrombin or homogeneouspost-translationally modified α-thrombin can be applied to a surface ina subject. The formulation can be applied with a solution comprisingfibrinogen. The formulation may be used, for example, in hemostasis,tissue fixation, graft fixation, wound healing and anastomosis.

In yet another aspect, provided herein is a formulation comprisingpurified β-thrombin as disclosed herein. In some embodiments, thepurified β-thrombin is obtained by the methods disclosed herein. Theformulation comprising the purified β-thrombin can be applied to asurface in a subject. The formulation can be applied with a solutioncomprising fibrinogen. The formulation may be used, for example, inhemostasis, tissue fixation, graft fixation, wound healing andanastomosis.

The term “purified β-thrombin”, typically, refers to a β-thrombinpreparation obtained following isolation of the β-thrombin fromα-thrombin, γ-thrombin and/or another protein present in the startingthrombin comprising solution using an anion exchange chromatographymethod. By “isolated” it is generally meant, when referring to “isolatedhomogenous post-translationally modified α-thrombin” or “isolatedβ-thrombin”, that the indicated molecule or compound is separate anddiscrete from the whole organism with which the molecule or compound isfound in nature and/or is sufficiently free of other molecules so thatthe molecule can be used for its intended purpose.

A “pharmaceutically acceptable carrier or diluent” refers to reagents,compounds, materials, compositions, diluents that are compatible withthe constituents in the formulation and suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio. A pharmaceutically acceptable carriersuitable for use with the formulation disclosed herein can be a liquid,semi-solid and solid material. A carrier may be a sponge, film, plaster,surgical dressing or a bandage.

In another aspect, provided herein is a method for hemostatic treatment,sealing, graft fixation, wound healing, anti-adhesion and/or anastomosisin a subject in need, comprising applying to the subject an effectiveamount of a formulation according to the invention. The terms “atherapeutically effective amount” or “an effective amount” refer to thedose required to prevent or treat (relieve a symptom or all of thesymptoms) a disease, disorder or condition. The effective amount can bemeasured based on any change in the course of the disease in response tothe administration of the formulation. The effective dose can be changeddepending on the age and weight of the subject, the disease and itsseverity (e.g. early or advanced stage) and other factors which can berecognized by the skilled in the art.

In another aspect, provided herein is a method for screening compoundsfor their potential use in stabilizing thrombin activity in an aqueousliquid thrombin formulation, the method comprising the steps of:incubating test compounds with a solution comprising α-thrombin for agiven time; after the incubation, quantifying the α-thrombin and/or thedegradation polypeptides (e.g. β-thrombin and/or γ-thrombinpolypeptides) according to method disclosed herein; and identifying oneor more suitable test compounds which have a potential use instabilizing thrombin activity, wherein a suitable compound is a compoundthat maintains the α-thrombin content at a level of about 70% to about100% compared to the initial α-thrombin content and/or which reduces thelevel of degradation polypeptides to about 0% to about 30% as comparedto the level of degradation polypeptides in the absence of the testcompound.

“Stabilizing thrombin activity” refers to, for example, reducingthrombin autolytic activity. “Stabilizing thrombin activity” may alsorefer to maintaining thrombin activity when stored for more than oneday, e.g. at room temperature as an aqueous thrombin solution e.g. aconcentrated thrombin solution; more than two years at equal to or lessthan −18° C.; and/or more than one month at 2-8° C., withoutsignificantly compromising thrombin's biological activity towardsheterologous substrates, including the activity of conversion offibrinogen to fibrin. “Room temperature” is meant to include temperatureof about 20° C. to about 25° C., or 22° C. to about 25° C. “Thrombinactivity” is meant to include thrombin mediated conversion ofheterologous substrates, including proteins e.g. fibrinogen into fibrin,as well as the conversion of Factor VIII to Factor VIIIa, XI to XIa,XIII to XIIIa, and Factor V to Va. A “heterologous substrate” is asubstrate, preferably a protein substrate, other than thrombin. In someembodiments, the thrombin activity refers to conversion of fibrinogeninto fibrin.

The term “stabilizing” means, for example, maintaining the thrombinactivity/potency within the thrombin liquid formulation at a level ofabout 70% to about 100% (e.g. about 90 to 100%) compared to the initialthrombin activity.

In some embodiments the compound(s) inhibit autolysis of thrombin byabout 70% to about 100%, about 70% to about 95%, about 70% to about 90%,or about 70% to about 80%, and retains about 70% to about 100%, about70% to about 95%, about 70% to about 90%, or about 70% to about 80%thrombin biological activity.

The term “test compounds” or “test substance” is a chemically definedcompound or mixture of compounds whose ability to stabilize thrombin isdefined by the methods of the invention. These compounds or mixtures ofcompounds can be any excipient(s)/stabilizers known in the art such asdescribed in Dave A. Parkins and Ulla T. Lashmar “The formulation ofbiopharmaceutical products”. PSTT Vol. 3, No. 4 April 2000.

The term “initial α-thrombin content” refers, for example, to theactivity of thrombin towards fibrinogen measured in a thrombin liquidformulation immediately after thawing a frozen thrombin formulation;immediately after reconstituting thrombin powder; and/or before storageof liquid thrombin under conditions that allow self-degradation (e.g.more than two years storage at equal or less than −18° C.; more than onemonth storage at 2-8° C.; and/or more than 1 day at room temperaturee.g. at concentrations of 800 IU/ml to 10,000 IU/ml thrombin or more).

In some embodiments, the incubation time is more than one day (e.g. atroom temperature) as an aqueous thrombin solution e.g. a concentratedthrombin solution; more than two years at equal to or less than −18° C.;and/or more than one month at 2-8° C.

The term “degradation polypeptides” refers to β-thrombin and/orγ-thrombin polypeptide.

The term “surface” may refer to an external surface of the skin that canbe seen by unaided vision and to a surface of an internal body partwhich is a part of the internal anatomy of an organism. Externalsurfaces include, but are not limited to, the skin of the face, throat,scalp, chest, back, ears, neck, hand, elbow, hip, knee, and other skinsites. Examples of internal body parts include, but are not limited to,body cavity or anatomical opening that are exposed to the externalenvironment and internal organs such as the nostrils; the lips; theears; the genital area, including the uterus, vagina and ovaries; thelungs; the anus; the spleen; the liver; and the cardiac muscle. Thesurface can be a bleeding or a non-bleeding site.

The formulations and kits disclosed herein can be used internally andexternally, for tissue and organ graft fixation, for sealing a surgicalwound, in vascular surgery including providing hemostasis, foranti-adhesion and for anastomosis such as arterial, gastrointestinal andtracheal anastomosis.

A “subject” as used herein, includes humans and animals of mammalianorigin. In one embodiment, a subject is a surgery patient or a woundedpatient.

The purified α-thrombin and/or the purified β-thrombin can be used inhemostatic products. The α-thrombin or β-thrombin can be used incombination with fibrinogen to form fibrin sealant.

The fibrinogen can be prepared from initial blood composition. The bloodcomposition can be whole blood or blood fractions, i.e. a product ofwhole blood such as plasma. In one embodiment of the invention, thefibrinogen component is comprised from a biologically active component(BAC) which is a solution of proteins derived from blood plasma whichcan further comprise tranexamic acid and/or stabilizers such asarginine, lysine, their pharmaceutically acceptable salts, or mixturesthereof. BAC can be derived from cryoprecipitate, in particularconcentrated cryoprecipitate.

The term “cryoprecipitate” refers to a blood component which is obtainedfrom frozen plasma prepared from whole blood. A cryoprecipitate can beobtained when frozen plasma is thawed in the cold, typically at atemperature of 0-4° C., resulting in the formation of precipitate thatcontains fibrinogen and Factor XIII. The precipitate can be collected,for example by centrifugation and dissolved in a suitable buffer such asa buffer containing 120 mM sodium chloride, 10 mM trisodium citrate, 120mM glycine, 95 mM arginine hydrochloride. The solution of BAC maycomprise further Factor VIII, fibronectin, von Willebrand factor (vWF),vitronectin, etc. for example as described in U.S. Pat. No. 6,121,232and WO9833533. Preferably, the composition of BAC can comprisestabilizers such as tranexamic acid and arginine hydrochloride.Typically, the amount of fibrinogen in BAC is in the range of from about40 to about 60 mg/ml. The amount of tranexamic acid in the solution ofBAC can be from about 80 to about 110 mg/ml. The amount of argininehydrochloride can be from about 15 to about 25 mg/ml.

Optionally, the solution is buffered to a physiological compatible pHvalue. The buffer can be composed of glycine, sodium citrate, sodiumchloride, calcium chloride and water for injection as a vehicle. Glycinecan be present in the composition in the amount of from about 6 to about10 mg/ml, the sodium citrate can be in the range of from about 1 toabout 5 mg/ml, sodium chloride can be in the range of from about 5 toabout 9 mg/ml and calcium chloride can be in the concentration of about0.1-0.2 mg/ml.

In another embodiment, the concentration of plasminogen and plasmin inthe BAC composition is lowered to equal or less than 15 μg/ml like forexample 5 μg/ml or less plasminogen using a method as described in U.S.Pat. No. 7,125,569, EP 1,390,485 and WO02095019. In another embodimentof the invention, when the concentration of plasminogen and plasmin inthe BAC composition is lowered, the composition does not containtranexamic acid or aprotinin. The fibrinogen solution may be the BAC2component (from EVICEL®) or any other fibrinogen containing solution,such as purified recombinant fibrinogen or cryoprecipitate produced fromhuman plasma.

Fibrinogen can be autologous, human including pooled plasma, or ofnon-human source. It is also possible that the fibrinogen is prepared byrecombinant methods or can be chemically modified.

While the following examples demonstrate certain embodiments of theinvention, they are not to be interpreted as limiting the scope of theinvention, but rather as contributing to a complete description of theinvention.

EXAMPLES

For all examples, herein, the following terms are used:

A “thrombin solution” refers to a solution of thrombin at about 3000IU/ml thrombin, a total protein concentration of about 1 mg/ml, in 20 mMsodium acetate at pH 6.9-7.1.

A “thrombin formulation” or “formulated thrombin” refers to a formulatedthrombin drug product EVITHROM® Thrombin, Topical (Human) (ETHICON,Inc.) or the thrombin component of EVICEL® Fibrin Sealant (ETHICON,Inc.), with a thrombin activity in the range of 800-1200 IU/ml, a totalprotein concentration of about 5.7-6.5 mg/ml, and 5.0 to 6.5 mg/ml humanserum albumin (HSA) at pH 6.9-7.1. The HSA used for the formulation ofThrombin includes a stabilizer, acetyltryptophan.

In all experiments below, the thrombin solution was used as a “controlsample” which comprises thrombin degradation polypeptides since thethrombin present is not formulated (e.g. does not comprise stabilizers)and highly concentrated (about 3000 IU/ml) and therefore the thrombin isprone to faster degradation (compared to the thrombin present in the“thrombin formulation”).

In the following Examples, tools were assessed for their ability toprovide separation between α-thrombin, its degradation polypeptides and,if present, HSA, and to quantify α-thrombin and its degradationpolypeptides.

In general, “good separation” is considered a “baseline resolution”between the peaks. “Baseline resolution” means an efficient separationof the analytes, in which the peaks detected as representative ofelution of the analytes do not overlap; that is, the detector responsereturns to the base line level between the peaks.

“Sufficient separation”—a clear distinction between the eluting peaksappears, however, the detector response does not fully return to thebase line level between the peaks.

Insufficient separation is considered—when overlaps of peaks appear inthe chromatogram.

Unless noted with values, the resolution/separation level was visuallyevaluated. Where numerical values are listed in the Examples below, theresolution (R_(s)), the extent to which a chromatographic columnseparates components from each other, is mathematically defined asfollows: resolution is the difference between the peak retention timesof a selected peak and the peak preceding it multiplied by a constant of1.18, then divided by the sum of the peak widths at 50% of peak height.

A resolution level of equal to or above 2 is considered a “baselineresolution” and therefore shows good separation and allows goodquantitation of the peaks. A resolution of equal to or above 1.5 (andlower than 2) is considered “sufficient separation” which enablesseparation and quantitation.

With regards to the chromatographic method efficacy, the terms“separation” and “resolution” are used interchangeably.

Example 1: Reverse-Phase High-Performance Liquid Chromatography(RP-HPLC) of HSA, Thrombin Solution and Formulated Thrombin

A standard procedure for separating proteins and fragments thereof isthe employment of HPLC devices in reverse phase mode. The basicprinciple of the RP-HPLC method is a device, consisting of a dual pump,a polar column, and a detector. The proteins are injected into thedevice and get retained on the column. Upon increasing the concentrationof organic solvents, the proteins and peptides retained on the columnare released from the column and elute into the detector, where aresponse is received based on the amount of proteins eluted at the giventime.

In the following Example, a RP-HPLC with a C4 column (Phenomenex,Jupiter, 00G-4167-B0, 4.6×250 mm) was evaluated as a tool to separatebetween α-thrombin, its degradation polypeptides and, if present, HSA,and to quantify α-thrombin and its degradation polypeptides.

The HPLC analysis was carried out using a Waters Alliance separationmodule, e2695 with a 100 μL injection loop; a photodiode array (PDA)detector, 2998 (scanning between A_(190 nm) to A_(450 nm)) was used withan integral Waters column oven at 50° C.

The organic solvents/solutions used for separation were:

Buffer A: HPLC grade water+0.1% (v/v) trifluoroacetic acid (TFA);

Buffer B: acetonitrile+0.1% (v/v) trifluoroacetic acid (TFA).

Different solution gradients (Buffer A and Buffer B ratios over time)were evaluated.

The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL HPLC gradewater as a blank sample.

In all the experiments, the different injection volumes were based onthe fact that thrombin in the thrombin solution was more concentrated ascompared to the formulated thrombin.

Prior to injection, all samples were filtered through a 0.45 μmPolyvinylidene difluoride (PVDF) membrane (Millipore, to filter outlarger particles e.g. aggregates).

The samples were stored at 10° C. in an integral sample compartmentuntil injected into the HPLC.

FIG. 1 shows a zoom-in view of a representative chromatogram in theregion of the eluting peaks.

In all Figs. the sample depictions is shown from top to bottom based onthe beginning of the chromatogram, the sample injected (from top tobottom) is listed on the chromatogram. The runs of the different samplesare shown in one figure as stacked overlays.

Although there was separation between the main peaks of HSA andthrombin, overall not enough separation was achieved. The peaks elutedfrom the column were too close to allow reliable separation and/orquantitation of the peaks.

Additional experiments were carried out where conditions includingtemperature, column chemistry (different tested RP columns are listedbelow), mobile phase chemistry (such as methanol) and gradients werealtered, yet the resolution between α-thrombin and its degradationpolypeptides and/or other proteins e.g. HSA did not improve.

The following additional RP columns were tested: Cosmosil C4, 5 μm, 300A, 4.6×250 mm; Sepax BioC18, 3 μm, 300 A, 4.6×150 mm; LiChroCART, 5 μm,300 A, 4×250 mm; Sepax C8, 5 μm, 300 A, 4×250 mm; Waters XBridge C4, 3.5μm, 300 A, 4×250 mm for the separation and quantitation as mentionedabove.

Therefore it was concluded that, RP-HPLC is not an appropriate tool if a“one-step” or “single column separation” and/or quantitation ofα-thrombin in the presence of degradation polypeptides and/or HSA isdesired.

Example 2: Anion Exchange High-Performance Liquid Chromatography(AEX-HPLC) and Elution Using a Linear Salt Gradient and pH 8.0

A standard procedure for separating proteins and fragments thereof isthe employment of HPLC devices in anion exchange mode. The basicprinciple of the AEX-HPLC method is a device, consisting of a dual pump,a polar column, and a detector. The proteins are injected into thedevice and are retained on the column. Upon changing the solventcharacteristics (e.g. salt concentration, pH), the proteins and peptidesretained on the column are released from the column and elute into thedetector, where a response is received based on the amount of proteinseluted at the given time.

In this experiment, HPLC analysis using an anion exchange column wasevaluated as a tool to separate between α-thrombin, its degradationpolypeptides and, if present, HSA and to quantify α-thrombin and itsdegradation polypeptides.

AEX-HPLC analysis was carried out using a Waters Alliance separationmodule, e2695 with a 100 μL injection loop; a PDA detector was used atA_(220nm) and A_(280nm), and an integral Waters column oven at 25° C.The column used was a Sepax 403NP5-4625 (Sepax Proteomix SAX-NP5 NP4.6×250 mm 403NP5-4625). The column (4.6 mm width and 250 mm in length)is based on 5 μm polymer beads. The beads have quaternary ammoniumchemistry and are non-porous, mono-disperse particles.

For elution from the AEX-HPLC a linear salt gradient between Buffer A:20 mM Tris pH 8.0 in HPLC grade water; and Buffer B: 20 mM Tris pH 8.0and 1 M NaCl in HPLC grade water were used.

The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL Buffer A as ablank sample.

Prior to injection, all samples were filtered through a 4 mm syringefilter with 0.45 μm pore size PVDF membrane. The samples were stored at10° C. in an integral sample compartment until injected into the HPLC.The run time was 37 minutes; the flow rate used was 0.8 mL/min and thepressure was about 2600 psi.

FIG. 2 shows a zoom-in view of a representative chromatogram in theregion of the eluting peaks.

The results show that AEX-HPLC with elution buffer at pH 8.0 and linearsalt gradient to 1 M NaCl did not provide sufficient separation and/orallow reliable quantitation. The peaks eluted from the column were tooclose to each other, the resolution was not sufficient.

Example 3: AEX-HPLC and Elution Using a Linear Salt Gradient and pH 6.0

The preceding Example showed that at pH 8.0 and a linear salt gradient,the separation between α-thrombin, its degradation polypeptides and HSAwas limited.

In this Example, elution using a phosphate buffer at pH 6.0 with anincreasing gradient to 1 M NaCl was evaluated using the column, deviceand experimental setup as described in Example 2.

For elution from the AEX-HPLC a linear salt gradient between Buffer A:20 mM phosphate buffer pH 6.0 in HPLC grade water; and Buffer B: 20 mMphosphate buffer pH 6.0 and 1 M NaCl in HPLC grade water were used.

The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL Buffer A as ablank sample.

FIG. 3 shows a representative chromatogram obtained for the differentsamples.

The results show that AEX-HPLC with elution buffer pH 6.0 and saltgradient to 1 M NaCl did not provide sufficient separation and/or allowreliable quantitation.

Acetyltryptophan seen in the chromatogram is a stabilizer present in theHSA formulation.

Example 4: AEX-HPLC and Elution Using a Linear Salt Gradient and pH 7.5

The preceding Examples showed that the separation was limited using anelution buffer at pH 6.0 (Example 3) and 8.0 (Example 2), and thereforean elution buffer at pH 7.5 was tested.

HPLC analysis and conditions were carried out as described in Example 2.Elution was carried out using a Tris buffer at pH 7.5 with an increasinglinear gradient of NaCl. The Buffers used were: Buffer A: 20 mM Tris pH7.5 in HPLC grade water; and Buffer B: 20 mM Tris pH 7.5 and 1 M NaCl.

The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL Buffer A as ablank sample. Results are shown in FIG. 4 (zoom-in view). The resultsshow that AEX-HPLC with elution buffer pH 7.5 and salt gradient to 1 MNaCl did not provide enough separation and/or allow reliablequantitation.

Example 5: AEX-HPLC and Elution Using a Linear NaNO₃ Salt Gradient andpH 8.0

As an alternative to NaCl, NaNO₃ (sodium nitrate) was evaluated for itsability to separate thrombin degradation polypeptide from α-thrombin andfrom the remaining proteins in solution e.g. HSA.

A linear salt gradient was evaluated between Buffer A: 20 mM Tris pH 8.0in HPLC grade water; and Buffer B: 20 mM Tris pH 8.0/1 M NaNO₃. Thecolumn, device and experimental setup were as described in Example 2.

The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; and c) 100 μL 5 mg/ml HSA. Results are shown inFIG. 5. As mentioned above, thrombin solution contained degradationpolypeptides.

The results show that the use of NaNO₃ as an eluent did greatly increasethe separation between HSA, acetyltryptophan, and thrombin, however, notsufficient resolution was achieved between thrombin and its degradationpolypeptides.

Example 6: AEX-HPLC and Elution Using a Linear Gradient Between pH 9.1to pH 3.4

As an alternative to using a salt gradient to achieve separation betweenthe relevant peaks eluted from the AEX-HPLC resin, a pH gradient usingamine based buffers was evaluated. Due to the amine character of thebuffer, the detection was carried out at A_(280nm).

Buffers A and B contained: 20 mM piperazine (Sigma Aldrich, P45907), 20mM triethanolamine (Sigma Aldrich, T9534), 20 mM bis-tris propane (SigmaAldrich, B4679), and 20 mM 1-methylpiperazine (Sigma Aldrich, 13000-1).

The buffers were adjusted to pH 9.1 (Buffer A) and pH 3.4 (Buffer B) bytitration with HCl. Total run time was 46 minutes. In all experiments, alinear gradient was run between steps 2 and 3 (see Table 1 below)—therun time for the linear gradient was 20 minutes. During steps 2 and 3,the materials are eluted from the column.

The samples injected were: a) 30 μL thrombin solution; b) 100 μLformulated thrombin; c) 100 μL 5 mg/ml HSA; and d) 100 μL Buffer A as ablank sample.

Flow conditions and the ratio between the buffers are presented inTable 1. The pH of the eluting buffer is dependent on the ratio betweenBuffer A and B. In general, a typical HPLC run consists at least of thefollowing steps:

An equilibrated column is loaded with the material (time between steps 1and 2—“Loading”).

Following this step, the material is eluted from the column (betweensteps 2 and 3—“Linear Gradient”). This can be carried out isocratically(without changing the buffer composition as compared to the Loadingand/or equilibration steps) or through a gradient (changing one of thebuffer characteristic, e.g. salt concentration, polarity/pH). In thisexample, elution was carried out using a linear gradient.

In the next step, the column can be regenerated (between steps 3 and4—“Column regeneration”), meaning that the remaining materials are givenadditional time at the highest concentration of the changedcharacteristic (salt concentration, polarity, pH) in order to elute fromthe column any remaining material.

The last step (between steps 5 and 6—“Column equilibration”) is anequilibration step, to allow the column to return to the original statein which the column is suitable for an additional separation

The conditions, column and device were as described in Example 2.

TABLE 1 Gradient and flow conditions. Time Flow rate Step (min) (mL/min)% Buffer A % Buffer B 1 0.01 0.80 90.0 10.0 2 5.00 0.80 90.0 10.0 325.00 0.80 0.0 100.0 4 30.00 0.80 0.0 100.0 5 31.00 0.80 90.0 10.0 646.00 0.80 90.0 10.0 Between steps 1 and 2 - Loading - 5 minutes.Between steps 2 and 3 - Linear Gradient - 20 minutes. The increment ofBuffer B was 4.5% per minute. Between steps 3 and 4 - “Columnregeneration” - 5 minutes. Between steps 5 and 6 - “Columnequilibration”- 15 minutes.

In all the Tables below the steps are characterized and numbered in thesame manner.

FIG. 6 shows representative chromatograms of the samples injected. FIG.7 is a zoom-in view of the thrombin eluting region in the chromatogramfrom FIG. 6.

The results show that good resolution was obtained between HSA,acetyltryptophan, and thrombin. In the described conditions, severalthrombin peaks were obtained (best shown in FIG. 6). In the followingexamples additional parameters were examined to further enhance theresolution of the thrombin peaks.

Example 7: AEX-HPLC and Elution Using a Linear pH Gradient at DifferentFlow Rates

In order to obtain better separation between the different thrombinpeaks, different flow rates were evaluated while keeping the temperature(at 25° C. as in Examples 2-7) and the pH gradient constant.

Buffers A and B were the same as in Example 6. The program (see Table 2below) was operated four times. Each time at a different flow rate:0.25, 0.5, 0.75, and 1 mL/min.

The gradients evaluated are as shown in Table 2 below.

The injected sample was 30 μL thrombin solution for each tested flowrate.

TABLE 2 Gradient and flow conditions. Time Step (min) % Buffer A %Buffer B 1 0.01 90.0 10.0 2 5.00 90.0 10.0 3 25.00 0.0 100.0 4 30.00 0.0100.0 5 31.00 90.0 10.0 6 46.00 90.0 10.0

FIG. 8 shows a zoom-in view of the chromatograms of the flow screencarried out.

The separation (visually inspected) between HSA, acetyltryptophan andthrombin was unaffected by the increase in flow rate (data not shown).

It was shown (FIG. 8) that the resolution between α-thrombin and itsdegradation polypeptides increases with increasing flow rates. The bestresolution was achieved at a flow rate of 1.0 mL/min e.g. more peaks areobserved.

Example 8: AEX-HPLC and Elution Using a pH Gradient from 100% Buffer A

In this Example the effect of starting the AEX-HPLC method at a higherpH, as compared to the previous Examples, on the separation resolutionwas evaluated. For this purpose, a pH gradient with 100% Buffer A (seeTable 3) was used instead of 90% (as used in Table 2). As a control, thesame set of samples were run in the manner described in Table 3, onlythat in steps 1, 2, 5, and 6 the percentage of Buffer A was 90 and thepercentage of Buffer B was 10.

Buffers A and B were the same as in Example 6. Unless written otherwise,the experimental setup was the same as in Example 6.

The resolution between the peaks was visually evaluated. Table 3 showsthe gradient and flow rate conditions.

TABLE 3 Gradient and flow conditions. Run Time Flow Step (min) (mL/min)% Buffer A % Buffer B 1 0.01 1.00 100.0 0.0 2 5.00 1.00 100.0 0.0 327.00 1.00 0.0 100.0 4 32.00 1.00 0.0 100.0 5 33.00 1.00 100.0 0.0 648.00 1.00 100.0 0.0 The increment of Buffer B was 4.55% per minute.

The results (data not shown) showed that starting the gradient at ahigher pH yielded a better resolution for the thrombin peaks.Accordingly, eluting the proteins from the column with a wider pH rangewill result in a better separation between the peaks.

In the following examples a pH gradient with 100% Buffer A was used.

Example 9: AEX-HPLC and Elution Using a Linear pH Gradient at IncreasingGradient Run Times

In order to obtain better separation/resolution between the differentthrombin peaks, increasing gradients (i.e. the time increase was betweensteps 2 and 3), each by five minutes to 51, 56, and 61 minutes total runtime, were evaluated as compared to the run time in Example 6 (i.e. thetime between steps 2 and 3 increased from 20 to 25, 30 and 35 minutes).A run time of 46 minutes (as in Example 6) was also tested. Theresolution was measured between each peak to its preceding peak.

Unless written otherwise, the experimental setup was the same as inExample 8 using the parameters listed in Table 3.

A thrombin solution (30 μL) was injected. Buffers A and B are same as inExample 6. Tables 4, 5, 6 and 7 show the retention time and theresolution achieved at 46, 51, 56, and 61 minutes total run time,respectively. Retention time is the interval between the instant ofinjection and the detection of the peak apex (the most upper point ofthe peak) as representative of elution.

TABLE 4 The resolution of the thrombin peaks at 46 minutes total runtime. Retention Time Peak of the peaks Number (min) Resolution 1 13.0912 14.525 3.009959 3 15.736 2.475773 4 17.171 3.529876 5 18.173 3.2797466 19.055 3.217816 7 20.337 5.087464

TABLE 5 The resolution of the thrombin peaks at 51 minutes total runtime. Retention Time Peak of the peaks Number (min) Resolution 1 12.5142 14.313 3.293685 3 15.852 2.902742 4 17.598 4.012924 5 18.852 3.8933946 19.990 3.642576 7 21.557 5.218836e

TABLE 6 The resolution of the thrombin peaks at 56 minutes total runtime. Retention Time Peak of the peaks Number (min) Resolution 1 13.0182 15.130 3.116849 3 16.980 2.923051 4 18.025 1.818590 5 19.050 2.1885106 20.549 4.065546 7 21.929 3.775590 8 23.792 5.461505

TABLE 7 The resolution of the thrombin peaks at 61 minutes total runtime. Retention Time Peak of the peaks Number (min) Resolution 1 10.6822 13.480 5.056362 3 15.912 3.370500 4 18.064 3.068171 5 19.292 1.8463186 20.453 2.142551 7 22.197 4.254319 8 23.824 3.981067

The results show that at 56 and 61 minutes total run times (a gradientlength of 30 and 35 minutes), an additional peak eluting in a regiondistinct to the thrombin peaks was separated as compared to the shorterrun times.

Advantageously, in order to obtain an additional peak eluting in thedistinct thrombin region, a gradient length of higher than 25 minutesmay be used.

In the next Examples, a total run time of 56 minutes was used.

Example 10: The Effect of the Linear Gradient Slope on the SeparationResolution

Different linear slope gradients were evaluated for their ability toimprove separation of thrombin peaks (gradients used between steps 2 and3). The slope is impacted by the increment of the percentage of Buffer Bper minute. A lower increase of the percentage of Buffer B per minuteresults in a shallower slope as compared to a higher increase of thepercentage of Buffer B per minute, thereby affecting the elution profileof the proteins. Contrary to Example 8 in which the gradient wasimpacted by using a different starting pH, in this Example, the gradientwas impacted by incrementing the pH value at different rates per minute(the pH of the start and endpoint are equal in all samples).

Buffers A and B were the same as in Example 6. Unless written otherwise,the experimental setup was the same as in Example 6. A thrombin solution(30 μL) was injected. Buffer A (30 μL) was used as blank (not shown).

The percentages increase of Buffer B per minute evaluated were: 4.5%,4.25%, 4%, 3.75%, and 3.5%. For example, when a percentage of 4.5% perminute was used, following the first minute 4.5% Buffer B per minute wasobtained, following the second minute 9% Buffer B per minute wasobtained, following the third minute 13.5% Buffer B per minute wasobtained etc. up to 100% Buffer B per minute. At each minute Buffer Awas used to complete the total solution to 100%.

Typically, a shallower slope results in an increased run time. The runtimes were as follows: 48, 49.5, 51, 52.7, and 54.6 minutes,respectively to the listed Buffer B percentage.

FIG. 9 shows the chromatogram obtained for the different gradientsevaluated, a visual inspection was carried out to determine theseparation resolution.

The results show that all tested slopes showed satisfactory/sufficientseparation between thrombin peaks with an increment of 3.5% having thebest separation (seen in zoom-in view, data not shown).

Example 11: Identification of the Different Thrombin Peaks by Injectionof Commercial Standards in AEX-HPLC

In order to identify the thrombin peaks in the chromatogram, a thrombinsolution was run as in Example 7 in addition to α, β and γ thrombinstandards using a flow rate of 1.0 mL/min.

Standards (Haematological Industries; Human alpha-Thrombin, HTIHCT-0020, Human beta-Thrombin, HTI-0022, Human gamma-Thrombin, HTI-0021)were diluted to 0.3 mg/mL before injection. 30 μL thrombin solution, 100μL of each α-, β- and γ-standards were injected into the HPLC. Buffer Aused as blank.

Buffers A and B were the same as described in Example 7 and used in theprogram shown in Table 2.

FIG. 10 shows the overlaid chromatograms. Based on the peaks obtainedfor the standards, it was possible to identify the correlating peaks ofthe thrombin solution and thereby verify that separation betweenα-thrombin and its degradation polypeptides β and γ thrombin can beachieved. In addition, it was noted that the α-thrombin elutes asmultiple peaks in the chromatogram.

Example 12: Thrombin Peaks Identification Using Western Blot as aQualitative Tool

In the previous Example thrombin peaks identification was carried out byinjection of commercial α, β and γ thrombin standards in AEX-HPLC.

To corroborate the above results, in this Example thrombin peaks werecollected from an injected thrombin solution and further qualitativelyidentified by Western Blot against commercial standards (as in Example11) based on the known size of α-thrombin and its degradationpolypeptides, β- and γ-thrombin.

In order to obtain sufficient amounts of β and γ thrombin, a thrombinsolution was incubated under conditions that enhance auto-degradation ofthrombin such as overnight for at least 12 hours at room temperature(about 20-25° C.) before injection into the HPLC. The experimental setupwas as in Example 10, the 3.5% B/min increase was used. 60 μL of thesample (in tetraplicates) and 100 μL Buffer A were injected.

The distinct peaks (shown and identified in FIG. 11 and Table 8) werecollected from the four separate runs (due to the small protein amountpresent in each peak), pooled (according to visual identification andretention time) and lyophilized due to the large collection volume. Eachlyophilized pooled peak was reconstituted (in a lower volume of water ascompared to the initial volume due to the limitations in the possibleload volume of the SDS-PAGE). The resulting pooled samples wereseparated by SDS-PAGE, transferred onto a nitrocellulose sheet andimmune-blotted against polyclonal anti-α-thrombin (data not shown). Amixture of α, β, γ was used as control.

The peaks were identified based on the molecular weights of the bandsobtained in the Western Blot and by comparison to the standard α, β, γmix.

TABLE 8 Peaks collected following injection of a thrombin solution.Retention time Peak of the peak Number (min) Identification 1 15.80 to16.51 α-thrombin 2 17.20 to 18.20 α-thrombin 3 18.55 to 19.00 β-thrombin4 19.00 to 19.50 α-thrombin 5 20.00 to 20.53 β and γ-thrombin   5a 20.53to 20.7  β and γ-thrombin 6 21.00 to 21.22 α-thrombin 7 21.9unidentified 8 22.20 to 22.40 α-thrombin 9 unidentified 10  unidentified

The results obtained in the Western Blot show that degradationpolypeptides of thrombin elute in peaks 3, 5 and 5a. Peaks 1, 2, 4, 6,and 8 having similar molecular weight, were identified as α-thrombin.The relative area of the peaks labeled as “unidentified” were smallcompared to the “identified” peaks.

Without being bound by the mechanism, α-thrombin is separated intoseveral peaks in the HPLC-AEX system and probably corresponds to severalα-thrombin species differing in their net charge.

The results of Examples 11 and 12 show that advantageously completeseparation between α, β, γ-thrombin, α-thrombin species and HSA (data ofHSA separation is not shown in this Example) can be obtained using anAEX-HPLC linear pH gradient between 100% Buffer A to 100% Buffer B witha slope of 3.5% Buffer B per minute. Buffer compositions are asdescribed in Example 6.

Example 13: Identification of α-Thrombin Species Resolved by HPLC-AEX

The objective of the present Example was to characterize the multiplepeaks detected for α-thrombin in HPLC-AEX chromatography. It wasexplored if the different species of α-thrombin are due to differentpost translated modified α-thrombin forms. There are severalpost-translation modifications; glycosylation is one possibility. Sinceglycosylation affects the activity of proteins (Ricardo J. Sola and KaiGriebenow. “Glycosylation of Therapeutic Proteins: An Effective Strategyto Optimize Efficacy”. BioDrugs. 2010; 24(1): 9-21), the followingExample focuses on glycosylation.

Human α-thrombin has a single N-linked glycosylation site on its “heavychain”. It was explored a possibility that the α-thrombin resolved inHPLC-AEX chromatography correspond to α-thrombin containing differentsialylation levels on the N-linked glycosylation site i.e. variableamounts of N-acetylneuraminic acid (NANA) (sialic acid) in theglycosylation site.

For this purpose, a thrombin solution was subjected toN-acetylneuraminidase treatment according to manufacturer's instructions(Sigma Aldrich, N2876). N-acetylneuraminidase (NANase) is an enzymecapable of removing the NANA residues from the terminal end of glycans.By removal of these charged sugar residues, the overall charge of eachof the glycosylated proteins is brought to the same level.

In the next step, the NANase-treated thrombin solution was injected toan AEX-HPLC system as described in Example 12. Thrombin solution withouttreatment was injected as control.

The results (FIG. 12) show that treatment of thrombin with NANaseaffects the elution profile resulting in an overall shift of the peaksto the left side of the chromatogram (as compared to the un-treatedthrombin solution). Due to the loss of the negative charge of the sialicacid residue, the protein net charge of thrombin at a given pH isincreased, thereby causing an earlier elution from the column. In viewof these results, it can be concluded that the numerous peaks resultsfrom differences in NANA content.

Example 14: Purifying Homogenously Post-Translationally Modifiedα-Thrombin from a Proteinatious Solution

In the previous Examples it was found that α-thrombin can be resolvedinto distinct peaks containing different amounts NANA/sialylation level.

In this example, the purpose was to isolate a homogenous α-thrombinspecies containing a substantially identical profile of NANA usingAEX-HPLC. The following conditions were used:

The column used was a Sepax 403NP5-4625, width: 4.6×length: 250 mm as inExample 2. 30 μL of thrombin solution, 100 μL formulated thrombin and100 μL of Buffer A (not shown) were injected.

Elution of proteins from the resin was carried out using a pH gradientcomposed of 20 mM piperazine (Sigma Aldrich, P45907), 20 mMtriethanolamine (Sigma Aldrich, T9534), 20 mM bis-tris propane (SigmaAldrich, B4679), and 20 mM 1-methylpiperazine (Sigma Aldrich, 13000-1).The buffers were adjusted to pH 9.1 (Buffer A) and pH 3.4 (Buffer B).

A linear pH gradient, with an increment of 3.5% Buffer B per minute, andflow conditions as shown in Table 9 were used.

TABLE 9 Gradient and flow conditions. Run Time Flow Step (min) (mL/min)% Buffer A % Buffer B 1 0.01 1.00 100.0 0.0 2 5.00 1.00 100.0 0.0 333.60 1.00 0.0 100.0 4 38.60 1.00 0.0 100.0 5 39.60 1.00 100.0 0.0 654.60 1.00 100.0 0.0

FIG. 13 shows the full length chromatogram of the two eluted thrombinsamples. FIG. 14 shows a zoom-in view of the α-thrombin species and thedegradation polypeptides eluting region.

For the formulated thrombin chromatogram it can be seen that completeseparation between HSA, several charged α-thrombin species (shown witharrows) and acetyltryptophan can be achieved.

For the thrombin solution chromatogram it can be seen that completeseparation between several charged α-thrombin species (shown witharrows) and degradation polypeptides can be achieved.

These results show that different homogenous α-thrombin species can beseparated from each other in a thrombin containing sample. Also, theresults show that the quality of the separation enables to purifyhomogenously post-translational modified α-thrombin from a proteinatioussolution and/or a solution comprising heterogeneously post-translationalmodified α-thrombin.

Example 15: Quantifying Homogenously Post-Translationally Modifiedα-Thrombin and Thrombin Degradation Polypeptides

The preceding examples show that α-thrombin peaks containing homogenouscontent of NANA can be well separated by the AEX-HPLC. Completeseparation of peaks allows quantitation of α, β, γ thrombin variants ina thrombin containing solution by calculating the integration of arelevant separated peak—see table 10. The conditions used in theAEX-HPLC were as described in the previous Example.

TABLE 10 Quantitation of the different thrombin variants. Retention Timeof the peak Identification (min) Area* Area (%)** α-thrombin 16.062219077 6.72 α-thrombin 17.698 2303906 70.65 β-thrombin 18.717 1193463.66 α-thrombin 19.230 273402 8.38 β and γ-thrombin 20.146 177410 5.44α-thrombin 21.000 62339 1.91 unidentified 21.543 5349 0.16 α-thrombin22.157 87076 2.67 unidentified 24.008 8456 0.26 unidentified 24.939 45560.14 *Area refers to the integrated area under the peak calculated bythe software. **The relative area from the total calculated peak area.

It was shown that quantification of all α-thrombin species and of thedegradation polypeptide was obtained.

The method can advantageously also be used to quantitate the amount ofα-thrombin from all proteins present in the solution and/or forscreening of suitable formulation.

Also, the results show that one type of β-thrombin can be purified andquantified using the method of the invention.

Although various embodiments have been described herein, manymodifications and variations to those embodiments may be implemented.Also, where materials are disclosed for certain components, othermaterials may be used. The foregoing description and following claimsare intended to cover all such modification and variations.

Any patent, publication, or other disclosure material, in whole or inpart, that is said to be incorporated by reference herein isincorporated herein only to the extent that the incorporated materialsdoes not conflict with existing definitions, statements, or otherdisclosure material set forth in this disclosure. As such, and to theextent necessary, the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.

Section headings are used herein to ease understanding of thespecification and should not be construed as necessarily limiting.

The invention claimed is:
 1. A one-step chromatographic method forquantifying α-thrombin in a solution comprising the α-thrombin, anotherprotein included in the solution for stabilization, and optionally anα-thrombin degradation polypeptide, wherein the ratio of thrombin to theanother protein in the solution is in the range of about 1 IU:10 mg toabout 1 IU:40 mg, the method comprising: contacting the solution with ananion exchanger; and separating the α-thrombin from the another protein,and from the optionally α-thrombin degradation polypeptide on anionexchange chromatography by differential elution conditions; andquantifying the α-thrombin.
 2. The method of claim 1, furtherquantifying one or more degradation polypeptides.
 3. The method of claim1, wherein the separated α-thrombin is a homogenous posttranslationallymodified α-thrombin, thereby quantifying homogenous post-translationallymodified α-thrombin.
 4. The method of claim 3, wherein the homogenouspost-translationally modified α-thrombin is a homogenous glycosylatedα-thrombin, thereby quantifying homogenous glycosylated α-thrombin. 5.The method of claim 4, wherein the separated homogenouspost-translationally modified α-thrombin is a homogenous sialylatedα-thrombin, thereby quantifying homogenous sialylated α-thrombin.
 6. Themethod of claim 1, wherein the solution comprises-another protein, andwherein the another protein is human serum albumin.
 7. A one-stepchromatographic method for quantifying homogenous posttranslationallymodified α-thrombin in a solution comprising heterogeneouspost-translationally modified α-thrombin and another protein included inthe solution for stabilization, wherein the ratio of thrombin to theanother protein in the solution is in the range of about 1 IU:10 mg toabout 1 IU:40 mg, the method comprising: contacting the solution with ananion exchanger; separating the homogenous post-translationally modifiedα-thrombin from the heterogeneous posttranslationally modifiedα-thrombin and from the another protein on anion exchange chromatographyby differential elution conditions; and quantifying the homogenouspost-translationally modified α-thrombin.
 8. The method of claim 7,wherein the solution further comprises at least one of an α-thrombindegradation polypeptide, and the method includes separating thehomogenous post-translationally modified α-thrombin also from theα-thrombin degradation polypeptide.
 9. The method of claim 1 or 7,wherein the differential elution conditions comprise a pH gradient. 10.The method of claim 9, wherein the pH gradient is generated by using aneluent comprising of an amine or a mixture of amines.
 11. The method ofclaim 1 or 7, wherein the anion exchanger is made of non-porousparticles.
 12. The method of claim 1 or 7, wherein the chromatographicmethod is an anion exchange High-Performance Liquid Chromatographymethod.